CA1198677A - Stable plurilamellar vesicles - Google Patents
Stable plurilamellar vesiclesInfo
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- CA1198677A CA1198677A CA000424498A CA424498A CA1198677A CA 1198677 A CA1198677 A CA 1198677A CA 000424498 A CA000424498 A CA 000424498A CA 424498 A CA424498 A CA 424498A CA 1198677 A CA1198677 A CA 1198677A
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- splvs
- entrapped
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Abstract
ABSTRACT
A new and substantially improved type of lipid vesicle, called stable plurilamellar vesicles (SPLVs), are described, as well as the process for making the same.
SPLVs are stable during storage and can be used in vivo for the sustained release of compounds and in the treatment of disease.
A new and substantially improved type of lipid vesicle, called stable plurilamellar vesicles (SPLVs), are described, as well as the process for making the same.
SPLVs are stable during storage and can be used in vivo for the sustained release of compounds and in the treatment of disease.
Description
1. FIELD OF THE INV:~:NTION
This invention relates to liposomes ~nd their uses as carriers in delivery systems. More ~pecifically~
it relates to a new type o:E lipid vesicle havirlg uni4ue 5 properties which confer special advantages such as increased stab.ility and high entrapment efficiencyr The compositions and methods described herein have a wide range of applicability to fields such as carrier systems and targeted delivery systems. The practice of the present invention is demonstrated herein by way of example for the treatment of brucellosis, the treatment of ocular infections, and the ~reatme~ of lymphocytic meningitis virus infections.
~' ~s ,-~8~
This invention relates to liposomes ~nd their uses as carriers in delivery systems. More ~pecifically~
it relates to a new type o:E lipid vesicle havirlg uni4ue 5 properties which confer special advantages such as increased stab.ility and high entrapment efficiencyr The compositions and methods described herein have a wide range of applicability to fields such as carrier systems and targeted delivery systems. The practice of the present invention is demonstrated herein by way of example for the treatment of brucellosis, the treatment of ocular infections, and the ~reatme~ of lymphocytic meningitis virus infections.
~' ~s ,-~8~
2. BACKGROgND O~ T~E INYENTIO~
2~ lo LIPOSCMES
Liposomes are co~pletely closed bilayer membranes containing an entrapp~d aqueous phase~ Liposomes ~ay be any variety of unilamellar vesicles (possessing a single membrane bilayer) or multilamellar vesicles (onion-like structures characterized by concentric membrane bilayers 10 each separated from the next by a layer of water).
The original liposome preparations of Bangham et al. (1965, J. Mol. ~iol~ l3:238-252) involved ~uspending phospholipids in an organic solvent which wa~ then 15 evaporated to~dryness leaving a waxy deposit of phospholipid on the reaction vessel. Then an appropriate amvunt of aqueous phase was added, the mixture was allowed to ~swell~, and the resulting liposomes which consisted of multilamellar vesicles (hereinaf~er referred to as MLVs) 20 were di5persed by mechanical means~ The structure of the resulting membrane bilayer i~ such ~hat the hydrophobic (non-polar) "tailsn of the lipid orient toward the center of the bilaye~ while the hydrophilic (polar) ~heads"
orient towards the aqueous phaseO This technique provided 25 the basis for the development of the small sonicate~
unilamellar vesicles thereinaf~er referred ~o as SUVs~
described by Papahadjapoulos and ~iller (1967, Biochim.
Biophys~ Acta~ 135 624-638)o These "classical liposomes", however, had a number of drawbacks not the le~st of which 30 was a low volume of entrapped a~ueous space per mole of lipid and a restricted ability ~o encapsulate large macromolecules.
Efforts to increase the entrapped v~lume involved 35 first forming inverse ~icelles or liposome precursors, i.e~, vesicle~ con~aining an a~ueou~ pha~e surrounded by a ~onolayer of lipid molecules ~ri*nted 50 that the polar head groups are dixected t~wards ~he aqueous pha eO
Liposome precursors are formed by adding the ~queous 5 solution ~o be entrappe~ to a o~utio~ of polar lipid in an organic ~olven~ and sonica~ing. The liposome precursors sre then evaporated in ~he presence o~ excess lipido The resultant liposomes, consisting of an aqueous phase entrapped by a lipid bilayer are dispersed in 10 aqueous phase (see U0 S. Patent No. 4,224,179 issued September 23, 1980 to M~ Schneider~.
In another attPmpt to maximize ~he efficiency of entrapment Papahadjopoulos ~U. S0 Patent NoO 4~235 j871 15 issued Novemb~r 25, 1980~ desc~ibes a ~rever~e-phase evaporation proces~" for m~king oligolamella~ lipid vesicles also known as rever~e-phase evaporation vesicles (hereinafter referred to as REVs). According to this procedure, the aqueous ma~erial to be entrapped is added 20 ~o a mixture of polar lipid in an organic solventO Then a homogeneous water-in-oil type sf emulsion is formed and the organic solvent is evaporated until a gel is formed.
The gel is then converted to a ~uspension by dispersing the gel-like mixture in an a~ueous media. The REVs 25 produced consist mostly of unilamellar vesicles and some oligolamellar vesicles which are characterized by only a few concentric bilayers with a large internal aqueous space. Certain permeability properties of REVs were reported to be similar to those of MLVs and SWs (see 30 Szoka and Papahad~opoulos, 1978, ProcO Natl. Acad. ~ci~
.S~A. 75 4194-4198)~
Liposomes which entrap a variety of compounds can be prepared, however, stability of the liposomes during 35 storage is invariably limited~ This loss in stability 7~
results in leakage of the entrapped compound from the lipos~mes lnto ~he surr4un~ing media, and can also result in contamination o~ the lipo~om2 con~ents by permeation of materials from the surrounding media into the liposome 5 it~elf. ~s a result the ~torage li~e of traditional liposomes i5 very limited. Attempts to improve ~bility involved incorporating into tAe liposome membrane certain ~ubstances (hereinaftex called ~tabilizers~ whioh affect the physical properties of the lipid bilayers (e.~., 10 steroid gro~ps). ~owever, many of these substances are relatively expensive and the production of such liposomes is not cost effective.
In addition to the storage problems of 15 traditional liposomes a number of compounds canno~ be incorporated into these vesicles~ MLVs can only be prepared under conditions above the phase-transition temperature of the lipid membrane. This precludes t~e incorporation of heat labile molecules within lipssomes 20 that are composed of phospholipias which exhibit desirable properties but possess lc>ng and highly saturated si~e chains .
2.2. ~SES O~ LIPOSOMES
Application o~ lipos~mes to therapeutic uses is described in Liposomes: From Physical Structures To Therapeutic Applications, Knight~ ed. Elsevier, North-Holland Biomedical Press, 1981~ Much has been 30 written regarding the possibilities of using these membrane vesicles for drug delivery systems though a number of problems with such systems remain. See, for example, ~he disclosures in U. S. Patent No. 3,993,754 issued on November ~3, lg76, to Yneh-Erh Rahman and 35 Elizabeth A. Cerny, and Uu S. Patent NoO 4,145,~10 iss~ea on Mar~h 20, 1979, to Barry D~, ~ear~. In a liposome drug 367'7~
delivery ~ystem the medicament i8 entrapped during liposome formation and then administered to the patient to be treated~ The medicament may be ~oluble in water or in a non-polar solvent~ Typical ~f ~uch di~clo~ure~ are 5 UO S~ P~tent 4,235,871 issued November 25, lg80, to Papahadjopoulos and Szoka and U9 S~ Patent ~,22~179 issued September 23, 1980 to M. Schneider.
Some desirable features of drug delivery systems 10 are resistance to rapid clearance of the drug accompanied by ~ sustained release of the drug whicb will prolong the drug's action. This increases effectiveness of the drug and allows the use of fewer administrations. Some of the problems encountered in using liposome preparations in 15 vivo include .he following: (1) Liposome entrapped materials leak when the liposomes are incubated in body fluids. Thi~ has been attributed to the removal of the liposo~al phospholipids by plasma high density lipoproteins (HDL), or to the degradation of the liposome 20 membrane by phospholipases, among other reasons. A result of the degradation o~ the lipssomes in vivo is that almost all the liposomal contents are released in a short period of time, therefore, sustained release and resistance of the drug to clearance are not ~chieved. (2) On the other 25 hand, if a very stable liposome is used in vivo (i e. 9 liposomes which do not leak when incubated in body fluids), then the liposomal contents will not be releasea as needed. As a result~ these stable liposomes are ineffective as carriers of therapeutic substances ~n vivo 30 because the sustained release or the ability to release the liposomal contents when necessary is not accvmplished. However, if one is treating an intraoellular infection, the maintenanoe of stability in biolo~ical fluids until the point that the liposome is 35 internalixed by the infected cell~ is critical. ~3) The o8 -c:ost~effectiveness of the lip~s~me carrier~ used in delivery ~ystem O For examE~le~ an improved method for the chemotherapy of lei~hmanial in~.ections using liposome encapsulated anti leishmanial clrus has been reported by 5 Steck and Alving in U. S. Paten~. No. 4 ,186 ,183 i~ued on January 29, 19~0. The liposomes used in the che~otherapy contained a number of stabilizers whic:h increased the stabili~y of ~he lipc~somes in vivo~, E[owever,~ as previously mentioned, these stabilizers are expensive and 10 the production of liposo~nes containing these stabilizers is not cost~effective,. (4) Ultimately, the problem encountered in the use of liposomes as carriers in drug delivery systems is l:he inability to e~fect a cure of the disease being treated. In addition to ~he inability to 15 resi~t rapid clearance and to ef fect sustained release, a number of other explanations for he inability to cure diseases are pos~ible. For instance, i~ the liposomes are internalized into target cells or phagocytic cells (~
reticuloendothelial cells), they are ~leared from the 20 system rapidly, rendering the entrapped drug largely ineffective against diseases of involving cells other than the RES~ After phagocytosis, ~he liposomal contents are package~ within lysosomes of the phagocytic cell. Very often the degradative enzymes contained within the 25 lysosome will degrade the entrapped compound or render the compound inactive by altering its structure or cleaving the compound at its active site. Furthermore, the liposomes may not deliver a dose which is effective due to the low efficiency of entrapment of active compound into 30 the vesicles when prepared.
Liposomes have also been used by researchers as model membrane systems and have been employed as ~he ~target cell" in complement mediated immunoassays~
35 However, when used in such assays ~ it i~ important that ~g~
the lipo~ome membrane do~s not leak ~hen incubated in sera because these assays mea~ure the rel~ase of the liposome contents as a function of serum complement ~ctivation by immune complex formation inv~lving certain im~unoglobulin classes (e.~, IgM and certain IgG molecules~.
2~ lo LIPOSCMES
Liposomes are co~pletely closed bilayer membranes containing an entrapp~d aqueous phase~ Liposomes ~ay be any variety of unilamellar vesicles (possessing a single membrane bilayer) or multilamellar vesicles (onion-like structures characterized by concentric membrane bilayers 10 each separated from the next by a layer of water).
The original liposome preparations of Bangham et al. (1965, J. Mol. ~iol~ l3:238-252) involved ~uspending phospholipids in an organic solvent which wa~ then 15 evaporated to~dryness leaving a waxy deposit of phospholipid on the reaction vessel. Then an appropriate amvunt of aqueous phase was added, the mixture was allowed to ~swell~, and the resulting liposomes which consisted of multilamellar vesicles (hereinaf~er referred to as MLVs) 20 were di5persed by mechanical means~ The structure of the resulting membrane bilayer i~ such ~hat the hydrophobic (non-polar) "tailsn of the lipid orient toward the center of the bilaye~ while the hydrophilic (polar) ~heads"
orient towards the aqueous phaseO This technique provided 25 the basis for the development of the small sonicate~
unilamellar vesicles thereinaf~er referred ~o as SUVs~
described by Papahadjapoulos and ~iller (1967, Biochim.
Biophys~ Acta~ 135 624-638)o These "classical liposomes", however, had a number of drawbacks not the le~st of which 30 was a low volume of entrapped a~ueous space per mole of lipid and a restricted ability ~o encapsulate large macromolecules.
Efforts to increase the entrapped v~lume involved 35 first forming inverse ~icelles or liposome precursors, i.e~, vesicle~ con~aining an a~ueou~ pha~e surrounded by a ~onolayer of lipid molecules ~ri*nted 50 that the polar head groups are dixected t~wards ~he aqueous pha eO
Liposome precursors are formed by adding the ~queous 5 solution ~o be entrappe~ to a o~utio~ of polar lipid in an organic ~olven~ and sonica~ing. The liposome precursors sre then evaporated in ~he presence o~ excess lipido The resultant liposomes, consisting of an aqueous phase entrapped by a lipid bilayer are dispersed in 10 aqueous phase (see U0 S. Patent No. 4,224,179 issued September 23, 1980 to M~ Schneider~.
In another attPmpt to maximize ~he efficiency of entrapment Papahadjopoulos ~U. S0 Patent NoO 4~235 j871 15 issued Novemb~r 25, 1980~ desc~ibes a ~rever~e-phase evaporation proces~" for m~king oligolamella~ lipid vesicles also known as rever~e-phase evaporation vesicles (hereinafter referred to as REVs). According to this procedure, the aqueous ma~erial to be entrapped is added 20 ~o a mixture of polar lipid in an organic solventO Then a homogeneous water-in-oil type sf emulsion is formed and the organic solvent is evaporated until a gel is formed.
The gel is then converted to a ~uspension by dispersing the gel-like mixture in an a~ueous media. The REVs 25 produced consist mostly of unilamellar vesicles and some oligolamellar vesicles which are characterized by only a few concentric bilayers with a large internal aqueous space. Certain permeability properties of REVs were reported to be similar to those of MLVs and SWs (see 30 Szoka and Papahad~opoulos, 1978, ProcO Natl. Acad. ~ci~
.S~A. 75 4194-4198)~
Liposomes which entrap a variety of compounds can be prepared, however, stability of the liposomes during 35 storage is invariably limited~ This loss in stability 7~
results in leakage of the entrapped compound from the lipos~mes lnto ~he surr4un~ing media, and can also result in contamination o~ the lipo~om2 con~ents by permeation of materials from the surrounding media into the liposome 5 it~elf. ~s a result the ~torage li~e of traditional liposomes i5 very limited. Attempts to improve ~bility involved incorporating into tAe liposome membrane certain ~ubstances (hereinaftex called ~tabilizers~ whioh affect the physical properties of the lipid bilayers (e.~., 10 steroid gro~ps). ~owever, many of these substances are relatively expensive and the production of such liposomes is not cost effective.
In addition to the storage problems of 15 traditional liposomes a number of compounds canno~ be incorporated into these vesicles~ MLVs can only be prepared under conditions above the phase-transition temperature of the lipid membrane. This precludes t~e incorporation of heat labile molecules within lipssomes 20 that are composed of phospholipias which exhibit desirable properties but possess lc>ng and highly saturated si~e chains .
2.2. ~SES O~ LIPOSOMES
Application o~ lipos~mes to therapeutic uses is described in Liposomes: From Physical Structures To Therapeutic Applications, Knight~ ed. Elsevier, North-Holland Biomedical Press, 1981~ Much has been 30 written regarding the possibilities of using these membrane vesicles for drug delivery systems though a number of problems with such systems remain. See, for example, ~he disclosures in U. S. Patent No. 3,993,754 issued on November ~3, lg76, to Yneh-Erh Rahman and 35 Elizabeth A. Cerny, and Uu S. Patent NoO 4,145,~10 iss~ea on Mar~h 20, 1979, to Barry D~, ~ear~. In a liposome drug 367'7~
delivery ~ystem the medicament i8 entrapped during liposome formation and then administered to the patient to be treated~ The medicament may be ~oluble in water or in a non-polar solvent~ Typical ~f ~uch di~clo~ure~ are 5 UO S~ P~tent 4,235,871 issued November 25, lg80, to Papahadjopoulos and Szoka and U9 S~ Patent ~,22~179 issued September 23, 1980 to M. Schneider.
Some desirable features of drug delivery systems 10 are resistance to rapid clearance of the drug accompanied by ~ sustained release of the drug whicb will prolong the drug's action. This increases effectiveness of the drug and allows the use of fewer administrations. Some of the problems encountered in using liposome preparations in 15 vivo include .he following: (1) Liposome entrapped materials leak when the liposomes are incubated in body fluids. Thi~ has been attributed to the removal of the liposo~al phospholipids by plasma high density lipoproteins (HDL), or to the degradation of the liposome 20 membrane by phospholipases, among other reasons. A result of the degradation o~ the lipssomes in vivo is that almost all the liposomal contents are released in a short period of time, therefore, sustained release and resistance of the drug to clearance are not ~chieved. (2) On the other 25 hand, if a very stable liposome is used in vivo (i e. 9 liposomes which do not leak when incubated in body fluids), then the liposomal contents will not be releasea as needed. As a result~ these stable liposomes are ineffective as carriers of therapeutic substances ~n vivo 30 because the sustained release or the ability to release the liposomal contents when necessary is not accvmplished. However, if one is treating an intraoellular infection, the maintenanoe of stability in biolo~ical fluids until the point that the liposome is 35 internalixed by the infected cell~ is critical. ~3) The o8 -c:ost~effectiveness of the lip~s~me carrier~ used in delivery ~ystem O For examE~le~ an improved method for the chemotherapy of lei~hmanial in~.ections using liposome encapsulated anti leishmanial clrus has been reported by 5 Steck and Alving in U. S. Paten~. No. 4 ,186 ,183 i~ued on January 29, 19~0. The liposomes used in the che~otherapy contained a number of stabilizers whic:h increased the stabili~y of ~he lipc~somes in vivo~, E[owever,~ as previously mentioned, these stabilizers are expensive and 10 the production of liposo~nes containing these stabilizers is not cost~effective,. (4) Ultimately, the problem encountered in the use of liposomes as carriers in drug delivery systems is l:he inability to e~fect a cure of the disease being treated. In addition to ~he inability to 15 resi~t rapid clearance and to ef fect sustained release, a number of other explanations for he inability to cure diseases are pos~ible. For instance, i~ the liposomes are internalized into target cells or phagocytic cells (~
reticuloendothelial cells), they are ~leared from the 20 system rapidly, rendering the entrapped drug largely ineffective against diseases of involving cells other than the RES~ After phagocytosis, ~he liposomal contents are package~ within lysosomes of the phagocytic cell. Very often the degradative enzymes contained within the 25 lysosome will degrade the entrapped compound or render the compound inactive by altering its structure or cleaving the compound at its active site. Furthermore, the liposomes may not deliver a dose which is effective due to the low efficiency of entrapment of active compound into 30 the vesicles when prepared.
Liposomes have also been used by researchers as model membrane systems and have been employed as ~he ~target cell" in complement mediated immunoassays~
35 However, when used in such assays ~ it i~ important that ~g~
the lipo~ome membrane do~s not leak ~hen incubated in sera because these assays mea~ure the rel~ase of the liposome contents as a function of serum complement ~ctivation by immune complex formation inv~lving certain im~unoglobulin classes (e.~, IgM and certain IgG molecules~.
3 . SVMI~ARY OF i:'ElE INVENTION
This invention presents a new and ~ubstantially improved type of lipid vesicles which hereinafter will be referred to as stable plurilamellar vesicles (SPLVs).
Aside from being ~tructurally different than multilamellar vesicles (MLVs), SPLVs are also prepared differently than MLVs, possess unique properties when compared to ~LVs~ and 15 present a v~riety of different advantages when compared to ~uch MLV~. As a re~ult of these di~ferences, SPLV~
overcome many of the problems presented by conventional lipid vesicles heretofore available.
A heterogeneous mixture of lipid vesicles is realized when SPLVs are synthesized. Evidence indicates that the lipids in the SPLVs are organi~ed in a novel ~upramolecular ~tructure. Many of the lipid vesicles possess a high number of bilayers, occasionally as high as 25 one hundred layers~ It may be possible that this high degree of layering contributes to many o~ the surprising properties possessed by SPLVs, although the explanations are theoretical.
The properties of SPLVs include~ (l) the ability to cure certain diseases which other methodologies cannot cure; (2) greatly increased stability of the SPLVs during storage in buffer; (3) the increased ability of SPLVs to withstand harsh physiologic environments; (4~ the 35 entrapment of materials at a high efficiency; (5) the '7~7 -ln-abili y to ~tick to tis~ues and cells ~or prolonged periods o~ time; (6) ~he ability ~o release of en$rapped materials ~lowly in body fluids; (7) ~he deli~ery and ultimate dispersal of the lipos~mal con~en~ throughout 5 the cytosol of ~he target cell (8) improved cost-effectivene~s in preparation; and (9~ rel2a~e of compounds in their bioactive forms in vivo.
Due ~o the unique properties o~ SPLVs they are 10 particularly u~eful as carriers in delivery systems ln v _ because th2y are resistant to clearance and are cap~ble of sustained release. Methods for the use o~
SPLVs for the delivery of bioactive compounds in vivo and the treatment of pathologies, ~uch as infections, are described~
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 graphically demonstrates the difference in 20 membrane ~tability (as reflected by % leakage) between MLVs and SPLVs treated with varyin~ concentrations of urea.
~ IG. 2 graphically represents the retention of both the lipid and aqueous phases of SPLVs in eyelid 25 tissues of mice, and the sustained release of gentamycin from the SPLVs ln vivo.
FIG. 3 represents the electron spin resonance absorption spectrum of SPLVs (A) compared to that of MLVs (B).
~ IG. 4 graphically demonstrates the dif~erence in the ability of ascorbate to reduce doxyl ~pin probes in SPLVs and in MLVs.
67~
. 5 graphically represer~ he eff~ctiveness s)f a two s~age treatanen'c of Brucella canis infections in mice u~ing SPLV-entrapped ~treptolDycin hased on B. canis recoverable from ~ple~ns of irlfected mice.
~ IG. 6 graphically represen~ the effectiveness of a two stage ~reatment of B. canis infections in mice using SPLV-entrapped ~treptomycin based on B. canls recoverable from organs of infected mice.
1~
FIG. 7 graphically represents the effectiveness of a two stage treatment of Brucella abortus in guinea pigs using SPLY-entrapped streptomycin.
5. DETAILEr) DE:SCRIPTION OF T}IE INVENTIO~
5 . l . PREPAR~TION OF SPLVS
SPLVs are prepared by a process which results in 20 a product unique from ~ny other liposome previously described.
SPLVs are lipid vesicles possessing from a few to over one hundred lipid bilayers. The membrane bilayer is 25 composed of a bimolecular layer o~ an amphipathic lipid in which the non-polar hydrophobic hydrocarbon ~tails"
point inward towards the center of the bilayer and the polar, hydrophilic "heads" point ~owards the aqueous phase. Occluded by ~he bilayers is an aqueous 30 compartment~ part of wich makes up the lumen of the vesicle, and part o~ which lie~ between adjacent layers.
Complexed with the lipid bilayers can b~ a variety of proteins, glyeoprotein~, glycolipids~ mucopolysacchariaes, and any other hydrophobic and/or amphipathic ~ubstance.
SPLVs are prepared as follow~: An amphipathic lipid or mixture of lipids is dissolYed in an organic fiolvent. ~any organic 601vent5 are ~uitable7 ~ut diethyl ether~ fluorina~ed hydrocarbons and mixture~ of 5 fluorinate~ hydrocarbons and e~her are preferred~ To ~his Eolution are added an aqueous pha~e ~nd the active ingredient to be entr~pped. Thi~ biphasic mixture is converted o SPLVs by emulsifying the aqueou~ material within ~he ~olvent while evaporating the ~olvent.
10 Evaporation ~an be accomplished during or after ~onication by any evaporative techni~ue9 e,~., evaporation by passing a stream of inert gas over the mixture, by heating~ or by vacuum. The volume of sol~rent use~ must exceed the aqueous volume by a ~ufficient amount ~o that the aqueous 15 material can be completely emulsified in the mixture. In practice/ a minimum of ro~ghly 3 volumes o~ solvent ~o 1 volume o~ aqueous phase may be used. In fact the ratio of solvent to aqueous phase can vary to up to 100 or mor~
volumes of solvent to 1 volume aqueous phase. The amount 20 of lipid mu~ be su~ficien~ so as tP exceed that amount needed to coat the emulsion droplets (about 40 mg of lipid per mQ of aqueous phase). The upper boundary is limited only by the practicality of cost-effç!ctiveness, but SPLVs can be made with 15 gm of lipid per mQ of aqueous phase~
The process produces lipid vesicles with different supermolecular organization than conventional liposomes. According to the present invention, the entire process can be performed at a temperature range of 4-60C
30 regardless of ~he phase transition tempera~ure of the lipid used. The advantage of this latter point is that heat labile products which have desirable properties, for example, easily denatured proteins, can be incorporated in SPLVs prepared from phospholipid ~uch as 35 distearoylphosphatidylcholine, b~lt can be formed into cs:~nventional lipo~omes only a~ ~emperatures above their phase-transi~ion-tempera~ure. The process usually allows more than 2096 of the available water ~oluble material to be encapsulated and mvre than 40% of the ~vailable lipid 5 soluble mater ial to be encapsula ed . With ~LVs t}-e entrapment of aqueous phase usually does not exceed 10%.
Most amphipathic lipids may be con~tituent~ of SPLVs. Suitable hydrophilic groups include but are not 10 limited 1:o: phosphato, carboxylic, ~ulphato and amino groups. Suitable hydrophobic groups include but are not limited to: saturated and unsatura~ed aliphatic hydro-carbon groups and aliphatic hydrocarbon groups substituted by at lea t one aromatic and/or cycloaliphatic ~roup. The preferred amphipathic compounds are phospholipids and closely related chemical structures. Examples of these include but are not limited to: lecithin, phosphatidyl-ethonolamine/ lysolecithin, lysophatidylethanolamine, pho~phatidylserine, phosphatidylinositol, sphingomyelin, 20 cardiolipin, phosphatidic acid and the cerebrosides.
Specific examples of suitable lipids u~eful in the production of SPLVs are phospholipids which include the natural lecithins (~ ~, egg lecithin or soybean lecithin) and synthetic lecithins, such as saturated synthetic lecithins ( ~ , dimyristoylphosphatidylcholine, or dipalmitoyl-phosphatidylcholine or distearoyl-phosphatidylcholine) and unsaturated synthetic lecithins ~ , dioloyl~phosphatidylchol ine or di 1 inoloyl-phosphatidylcholine. The SPLV bilayers can contain a 3~ steroid component such as cholesterol, coprostanol, cholestanol~ cholestane and the like. When using compounds with aci~ic hydrophilic groups (phosphato, sulfato, etc.) the obtained SPLVs will be anionic; with basic groups such as amino, cationic liposomes will be obtained; and with polyethylenoxy or glycol groups neutral liposome~ will be ob ained. The size of ~he S~V5 varies widely. The range extends from about 500 nm to about 10,000 nm ~10 mi rons) and usually abou~ 1,000 ~m to abo~t
This invention presents a new and ~ubstantially improved type of lipid vesicles which hereinafter will be referred to as stable plurilamellar vesicles (SPLVs).
Aside from being ~tructurally different than multilamellar vesicles (MLVs), SPLVs are also prepared differently than MLVs, possess unique properties when compared to ~LVs~ and 15 present a v~riety of different advantages when compared to ~uch MLV~. As a re~ult of these di~ferences, SPLV~
overcome many of the problems presented by conventional lipid vesicles heretofore available.
A heterogeneous mixture of lipid vesicles is realized when SPLVs are synthesized. Evidence indicates that the lipids in the SPLVs are organi~ed in a novel ~upramolecular ~tructure. Many of the lipid vesicles possess a high number of bilayers, occasionally as high as 25 one hundred layers~ It may be possible that this high degree of layering contributes to many o~ the surprising properties possessed by SPLVs, although the explanations are theoretical.
The properties of SPLVs include~ (l) the ability to cure certain diseases which other methodologies cannot cure; (2) greatly increased stability of the SPLVs during storage in buffer; (3) the increased ability of SPLVs to withstand harsh physiologic environments; (4~ the 35 entrapment of materials at a high efficiency; (5) the '7~7 -ln-abili y to ~tick to tis~ues and cells ~or prolonged periods o~ time; (6) ~he ability ~o release of en$rapped materials ~lowly in body fluids; (7) ~he deli~ery and ultimate dispersal of the lipos~mal con~en~ throughout 5 the cytosol of ~he target cell (8) improved cost-effectivene~s in preparation; and (9~ rel2a~e of compounds in their bioactive forms in vivo.
Due ~o the unique properties o~ SPLVs they are 10 particularly u~eful as carriers in delivery systems ln v _ because th2y are resistant to clearance and are cap~ble of sustained release. Methods for the use o~
SPLVs for the delivery of bioactive compounds in vivo and the treatment of pathologies, ~uch as infections, are described~
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 graphically demonstrates the difference in 20 membrane ~tability (as reflected by % leakage) between MLVs and SPLVs treated with varyin~ concentrations of urea.
~ IG. 2 graphically represents the retention of both the lipid and aqueous phases of SPLVs in eyelid 25 tissues of mice, and the sustained release of gentamycin from the SPLVs ln vivo.
FIG. 3 represents the electron spin resonance absorption spectrum of SPLVs (A) compared to that of MLVs (B).
~ IG. 4 graphically demonstrates the dif~erence in the ability of ascorbate to reduce doxyl ~pin probes in SPLVs and in MLVs.
67~
. 5 graphically represer~ he eff~ctiveness s)f a two s~age treatanen'c of Brucella canis infections in mice u~ing SPLV-entrapped ~treptolDycin hased on B. canis recoverable from ~ple~ns of irlfected mice.
~ IG. 6 graphically represen~ the effectiveness of a two stage ~reatment of B. canis infections in mice using SPLV-entrapped ~treptomycin based on B. canls recoverable from organs of infected mice.
1~
FIG. 7 graphically represents the effectiveness of a two stage treatment of Brucella abortus in guinea pigs using SPLY-entrapped streptomycin.
5. DETAILEr) DE:SCRIPTION OF T}IE INVENTIO~
5 . l . PREPAR~TION OF SPLVS
SPLVs are prepared by a process which results in 20 a product unique from ~ny other liposome previously described.
SPLVs are lipid vesicles possessing from a few to over one hundred lipid bilayers. The membrane bilayer is 25 composed of a bimolecular layer o~ an amphipathic lipid in which the non-polar hydrophobic hydrocarbon ~tails"
point inward towards the center of the bilayer and the polar, hydrophilic "heads" point ~owards the aqueous phase. Occluded by ~he bilayers is an aqueous 30 compartment~ part of wich makes up the lumen of the vesicle, and part o~ which lie~ between adjacent layers.
Complexed with the lipid bilayers can b~ a variety of proteins, glyeoprotein~, glycolipids~ mucopolysacchariaes, and any other hydrophobic and/or amphipathic ~ubstance.
SPLVs are prepared as follow~: An amphipathic lipid or mixture of lipids is dissolYed in an organic fiolvent. ~any organic 601vent5 are ~uitable7 ~ut diethyl ether~ fluorina~ed hydrocarbons and mixture~ of 5 fluorinate~ hydrocarbons and e~her are preferred~ To ~his Eolution are added an aqueous pha~e ~nd the active ingredient to be entr~pped. Thi~ biphasic mixture is converted o SPLVs by emulsifying the aqueou~ material within ~he ~olvent while evaporating the ~olvent.
10 Evaporation ~an be accomplished during or after ~onication by any evaporative techni~ue9 e,~., evaporation by passing a stream of inert gas over the mixture, by heating~ or by vacuum. The volume of sol~rent use~ must exceed the aqueous volume by a ~ufficient amount ~o that the aqueous 15 material can be completely emulsified in the mixture. In practice/ a minimum of ro~ghly 3 volumes o~ solvent ~o 1 volume o~ aqueous phase may be used. In fact the ratio of solvent to aqueous phase can vary to up to 100 or mor~
volumes of solvent to 1 volume aqueous phase. The amount 20 of lipid mu~ be su~ficien~ so as tP exceed that amount needed to coat the emulsion droplets (about 40 mg of lipid per mQ of aqueous phase). The upper boundary is limited only by the practicality of cost-effç!ctiveness, but SPLVs can be made with 15 gm of lipid per mQ of aqueous phase~
The process produces lipid vesicles with different supermolecular organization than conventional liposomes. According to the present invention, the entire process can be performed at a temperature range of 4-60C
30 regardless of ~he phase transition tempera~ure of the lipid used. The advantage of this latter point is that heat labile products which have desirable properties, for example, easily denatured proteins, can be incorporated in SPLVs prepared from phospholipid ~uch as 35 distearoylphosphatidylcholine, b~lt can be formed into cs:~nventional lipo~omes only a~ ~emperatures above their phase-transi~ion-tempera~ure. The process usually allows more than 2096 of the available water ~oluble material to be encapsulated and mvre than 40% of the ~vailable lipid 5 soluble mater ial to be encapsula ed . With ~LVs t}-e entrapment of aqueous phase usually does not exceed 10%.
Most amphipathic lipids may be con~tituent~ of SPLVs. Suitable hydrophilic groups include but are not 10 limited 1:o: phosphato, carboxylic, ~ulphato and amino groups. Suitable hydrophobic groups include but are not limited to: saturated and unsatura~ed aliphatic hydro-carbon groups and aliphatic hydrocarbon groups substituted by at lea t one aromatic and/or cycloaliphatic ~roup. The preferred amphipathic compounds are phospholipids and closely related chemical structures. Examples of these include but are not limited to: lecithin, phosphatidyl-ethonolamine/ lysolecithin, lysophatidylethanolamine, pho~phatidylserine, phosphatidylinositol, sphingomyelin, 20 cardiolipin, phosphatidic acid and the cerebrosides.
Specific examples of suitable lipids u~eful in the production of SPLVs are phospholipids which include the natural lecithins (~ ~, egg lecithin or soybean lecithin) and synthetic lecithins, such as saturated synthetic lecithins ( ~ , dimyristoylphosphatidylcholine, or dipalmitoyl-phosphatidylcholine or distearoyl-phosphatidylcholine) and unsaturated synthetic lecithins ~ , dioloyl~phosphatidylchol ine or di 1 inoloyl-phosphatidylcholine. The SPLV bilayers can contain a 3~ steroid component such as cholesterol, coprostanol, cholestanol~ cholestane and the like. When using compounds with aci~ic hydrophilic groups (phosphato, sulfato, etc.) the obtained SPLVs will be anionic; with basic groups such as amino, cationic liposomes will be obtained; and with polyethylenoxy or glycol groups neutral liposome~ will be ob ained. The size of ~he S~V5 varies widely. The range extends from about 500 nm to about 10,000 nm ~10 mi rons) and usually abou~ 1,000 ~m to abo~t
4~000 nm.
Virtually any bioactive compound can be entrapped wi~hin a SPLV (entrapped 1~ defined as ~n~rapment within ~he aquevus compartment or wi~hin the membrane bilayer).
Such compounds include but are not limited ~o nucleic 10 acids, polynucleotides, antibacterial compounds~ antiviral compounds, antifungal compounds, anti-parasitic compounds, tumoricidal compoundsl proteins, toxins, enzymes, hormones, neurotransmi~ters, glycoprotein~, i~munoglobulins, immunomodulators, dyes, radiolabels, 15 radio-opague compounds, fluorescen~ compounds, polysaccharides, cell receptor binding molecules J
anti-inflammatories, antiglaucomic agent~, mydriatic compounds, local anesthetics, etc.
The following is an example of the proportions that may be used in SPLV synthesis: SPLVs may be formed by adding 50 micromoles of phospholipid to 5 mQ of diethyl ether containing 5 micrograms of BHT
(~utylatedhydroxytoluene) and then adding 0.3 mQ of 25 aqueous phase containing the active substance to be encapsulated. The resultant solution which comprises the material to be entrapped and the entrapping lipid is sonicated while streaming an inert gas over the mixture thus removing mo~t of the solvent. This embodiment 30 produces particularly ~table SPLVs partially because of the incorporation of BRT into the ve~icles.
See also Lenk, et al., 1982, Eur. J. Biochem.
1 .475 482 which describes a process for making 35 liposome-encapsulated antibodies by sonica ing and evaporating a ~olution o~ chol~esterol and pho~phatidyl-choline in a ~nixture of s:hloroform and e~her wi~h aqueous phase added, but does not set forth the relative pr~portions of lipid to agueous phase.
Virtually any bioactive compound can be entrapped wi~hin a SPLV (entrapped 1~ defined as ~n~rapment within ~he aquevus compartment or wi~hin the membrane bilayer).
Such compounds include but are not limited ~o nucleic 10 acids, polynucleotides, antibacterial compounds~ antiviral compounds, antifungal compounds, anti-parasitic compounds, tumoricidal compoundsl proteins, toxins, enzymes, hormones, neurotransmi~ters, glycoprotein~, i~munoglobulins, immunomodulators, dyes, radiolabels, 15 radio-opague compounds, fluorescen~ compounds, polysaccharides, cell receptor binding molecules J
anti-inflammatories, antiglaucomic agent~, mydriatic compounds, local anesthetics, etc.
The following is an example of the proportions that may be used in SPLV synthesis: SPLVs may be formed by adding 50 micromoles of phospholipid to 5 mQ of diethyl ether containing 5 micrograms of BHT
(~utylatedhydroxytoluene) and then adding 0.3 mQ of 25 aqueous phase containing the active substance to be encapsulated. The resultant solution which comprises the material to be entrapped and the entrapping lipid is sonicated while streaming an inert gas over the mixture thus removing mo~t of the solvent. This embodiment 30 produces particularly ~table SPLVs partially because of the incorporation of BRT into the ve~icles.
See also Lenk, et al., 1982, Eur. J. Biochem.
1 .475 482 which describes a process for making 35 liposome-encapsulated antibodies by sonica ing and evaporating a ~olution o~ chol~esterol and pho~phatidyl-choline in a ~nixture of s:hloroform and e~her wi~h aqueous phase added, but does not set forth the relative pr~portions of lipid to agueous phase.
5 ~ 2~, C~ARACTERI 3~ATION OF SPLVS
SPLVs are ::learly distinct in their properties from liposomes with a single or several lamellae (e.~., 10 SUVs, ~nd REVs). Freeze-~racture electron microscopy indicates that SPLV prepara'tions are substantially îre~o of SWs and REVs, that is, less than 20% of the vesicles are unilamellar. They ar~7 however, indistinyuishable from MLVs by electron microscopic technique5 although many of 15 their physieal properties are different. Thus, the following detailed comparison is focused on di~;tinguishing SPLVs f r om MLVs .
S. 2.1. STABILITY OF SPLVS IN STORAGE
Stability o a lipid vesicle refers to the ability of the vesicle to sequester its occluded space from th~ external environment over a lvng period of time.
For a lipid vesicle to be useful it i~ paramount that it be stable in storage and handling. For ~ome applications, however, it is desirable that the vesicle leak its contents slowly when applied. For other applications it is desirable that the vesicle remain intact after administration until it reaches its desired site of 30 action. It will be seen that SPLVs demonstrate these desirable charac:~eristics, while P~LVs do not.
There are two factors that cause vesicles to leak. One is auto-oxidation oî the lipids whereby the 35 hydrocarbon chains form peroxides which destabilize the 7'7 bilayer~. ~his oxidation can be dra~tically slowed down by the addition of an ioxidants 8UCh as butylated hydroxy toluene ~BHT) to the vesicle preparation. Ve~icles can aiso le~k becau~e agent6 i~ the exterior environment disrupt the bilayer organiza$ion of the lipid8 ~uch that ~he lipids remain intact, but the membrane develops a pore.
Pre~arations of lipid vesicles are white in color when first msde. Upon au~o-oxida~ion, ~he preparation 10 becomes discolored (browni~h). A comparison of MLVs to SPLVs prepared using the same lipid and aqueous components reveals tha~ MLVs discolos wi~hin one to two week~ whereas SPLY~ remain white for at least two months. This is supported by thin layer chromatography of the constituent 15 lipi~s which ~howed degradation of ~he lipids in ~he MLVs but not of the lipids of ~he 5PLV~. When ~hese ve~icles are prepared by adding BHT as well as the other constituents~ then MLVs appear slightly discolored within one month whereas the SPLVs remain white and appear stable 20 for at least 6 months and longer.
When placed in a bu~fer containing isotonic saline a~ neu~ral p~, SPLVs containing antibiotic are ~table for more than four months, as demonstrated in 25 Table X. These data indicate that none of the antibiotic originally encapsulated withln the SPLVs leaked out in the period of the experiment.
Other evidence indicates that SPLVs are able to 30 ~equester an encapsulated agent from molecules as ~mall as calcium ions for more than 5iX months~ Arsenazo III is a dye which changes color from red to blue with the slightest amount of divalent cation present. By encapsulating the dye in SPLVs and adding calcium chloride 35 to the stora~e buffer it is possible to measure the >77 -;L7-TABLE I
5STABILITY OF EGG P~QSPHATIDYLC~OLINE
SPLV~ A~l~ER STORAG~ IN SEALED
CONT~INERS AT ~C ~OR 4-1/2 ~ONTHS a Initial Læakage Bioavailabillty Entrapped ~:ntrapment ~nto of Entrapped Drug % Supernatantb Drug (%) 10 Strept~mycin Sulfate 34.1 0 97 Spectinomycin37.2 0 84 Chloramphenicol 35.~ 0 89 ~5 Oxytetracycline 1~.8 0 gl Erythromycin 0 D 4 0 97 Sulfamerazine 6 . 3 0 93 20 a 5PLVs were prepared using 127 ~M egg phosphatidylcholine (EPC~ and 25 IIM drug. At the end of 4 1/2 months storage at 4C the SPLVs were separated from storage buffex by centrifugation.
Ser ial dilutions of the SPLV contents and ~h supernatant were applied to bacterial lawns in order to determine bioactivity as compared to standard dilutions of an~ibiotic.
b o indicates below detectable levels stabili~y of the vesicles by looking for a color change.
The color remains undetectably different from its original rolor for at least 6.5 months, demonstrating that neither has the dye leaked out nor the ion leaked in~
These experiments demonstrate that SPLVs are sufficiently stable to withstand storage and handling -~.8-proklem60 ~l~hough i~ is possible ~o make MLVs which are ~ta~le ~or this long, they mus~: be made from synthetic lipids such as DSPC and thus become prohibitively expen~ive e 5.2.2. STA~ILITY O~ SPLVS IN OTHER ENVIRONMENTS
Placing lipid vesicles in a medium which contains membrane perturbing agents is a way to probe different 10 molecular organizations, Depending on how ~he membrane is organized, different ve~icles will respond differently to such agents~
In the following experiments vesicle~ were 15 prepared which contained a radioactive tracer molecule (3H inulin) within the o~cluded a~ueous compartment~
Inulin, a polysaccharide, partition into ~he aqueous phase, and thus when radiolabeled may be u~ed to tr~ce the aqueous contents of lipid vesicles. After an appropriate 20 interval of exposure to a given agent, the vesicles were separated from the loedium by centrifugation, and the relative amount of radi~activity that escaped from the vesicles into the medium was determined. These results are reported in Table II; valu~s are expre~sed as pexcent 25 leaked, meaning ~he proportion of radioactive material in the surrounding medium relative ~o the starting amo~nt encapsulated in 'che vesicles.
SPLV~ are more stable than MLVs in hydrochloric 30 acid. Table II illl3strates that both MLVs ~nd SPLVs, when ~de from egg lecithin, are destabili~ed when exposed to 0.125 N hydrochloric acid for one hour. i~owever, it is no~eworthy that the SPLVs are considerably less ~usceptible to ~he acid than MLVs~ Presumably this 3~ different response reflects an intrinsic difference in the way the lipids in~eract with their environment.
7~
TABLE I I
STA:3ILITY OF SPLVS IN OTEIER E:NVIPcONM;~3TS
Incubating Mediuma 96 LEAKAGE
MLVs SPLVs :~ydr och lor ic Ac id 0.125M 90.5 55.2 ~ Urea lM 21.7 44.8 Guanidine OoSM 5~7 7~
1.0M 303 10.1 15 Ammon i um Ac e ta te 0.5M 27.0 67~0 1~0M 25.9 54.7-63.1 Serum 76 . 2 57 . 8 20 a Incubation time is 2 to 4 hours except incubation in HCl was f or 1 hour .
SPLVs also re~pond different:Ly than MLVs when exposed to urea (FIG. 1 and Table II3. Urea is a molecule with both a chaotropic effect (disrupts the structure of water) and a strong dipole moment. It i~ observed that SPLVs are f~r more susceptible to urea than they are to an osmotic agent such as sodium chloride at the same 30 concentration (FIG. 1). MLV~ do not leak significantly more in urea than they would in ~odium chloride. Althou~h the explanations for this different behavior are ~heore~ieal, it would ~ppear that 'che response is due to ~he dipole effec~, rather than a chaotropic property9 35 ~ince guanidine, a molecule similar to urea, does not destabilize SPLVs (Table II). Although guanidine is also -;2~-~trongly chaotxopic, it doe~ no~ po~ses~ a ~rong dipole moment.
SPLVs are also su&ceptible to ammonium acetate, 5 while MLVs are not (Table II). ~owever, nei~her ~mmonium ion (in ammonium chloride~ nor acetate ~in ~odium acetate) ~re par~icularly effective in ~ausing SP~Vs ~o destabilize. ThuS it would appear ~hat i~ is no~ the ivn itself, but the polarity of the ammonium acetate which is 10 responsible for inducing leakage.
Initially these results ~eem urpri~ing because SPLVs are much more stable ~han MLVs ~hen incubated in body fluids such as ~era or blood. ~owever a ~heoretical 15 explanation for these re~ults can be proposed tof course other explanations ~re possible)7 If the stability of the SPLV is due to the unique structure of its membrane bilayers ~uch that the polar groups of the membrane lipids are hydrated by a cloud of oriented water molecules, or 20 hydration ~hell, then it i~ possible that any agent which disrupts or interferes with such hydration shells would promote changes in structural membrane integrity, and therefore, leakage.
Independent of the theoretical explanations for ~he destabilization of SPLVs in urea are correct, the results serve to demonstrate characteristic differences between the structure of MLVs and SPLVs. This difference serves a very useful purpose in application. As described 30 infra~ SPLVs become slowly leaky when applied ~o the eye.
Presumably this desired slow release of contents is due to a similar destabilization of the SPLVs when expose~ to tear fluid~
SPLVs are more stable in serum than MLVs. Many applications of lipid vesicles include administering them JrJ
intraperitoneally~ such as for the treat~ent of brucellosisO To be effec~ive, ~he vesicles mus~ survive for a ~u$ficien~ ~ime ~o reach their desired target~
SPLVs ~nd MLVs, both made from l~gg lecithin, were exposed 5 to fetal bovine erum which con~ained active complement, (Table II). After 48 hours exposure at 37C, ~PLV~ are demonstrably more ~table than MLVs.
5.2.3. CHARACIERISTICS O~ SPLVS ADMINISTERED I~ VIVO
SPLVs demonstrate a number of characteristics which make them particularly suitable as carriers for delivery systems in v~vo:
(A) SPLVs are resistant to clearance. When SPLVs are administered to an or~anism both the lipi~
component and the entrapped ac ive ingredient are retaine~
in the t~ssues and by the cells to which they sre administered;
~ B) SPLVs can be engineered to provide sustained selease. The stability of SPLVs i5 "adjustable" in that SPLVs are very stable during storage and are stable in the presence of body fluids but when administered ~.n vivo a 25 slow leakage of the active ingredient permits the sustained release of the ac~ive ingredient;
(C) Because of the high level of entrapment ana stability when administered, effective doses of the active 30 ingredient are rele~sed; and (D) The production of SPLVs is very cost effective in that stability o~ ~he vesicles is achieYed without incorpora~ing expensive stabilizers in~o ~he 35 bilayers.
SPLVs are ::learly distinct in their properties from liposomes with a single or several lamellae (e.~., 10 SUVs, ~nd REVs). Freeze-~racture electron microscopy indicates that SPLV prepara'tions are substantially îre~o of SWs and REVs, that is, less than 20% of the vesicles are unilamellar. They ar~7 however, indistinyuishable from MLVs by electron microscopic technique5 although many of 15 their physieal properties are different. Thus, the following detailed comparison is focused on di~;tinguishing SPLVs f r om MLVs .
S. 2.1. STABILITY OF SPLVS IN STORAGE
Stability o a lipid vesicle refers to the ability of the vesicle to sequester its occluded space from th~ external environment over a lvng period of time.
For a lipid vesicle to be useful it i~ paramount that it be stable in storage and handling. For ~ome applications, however, it is desirable that the vesicle leak its contents slowly when applied. For other applications it is desirable that the vesicle remain intact after administration until it reaches its desired site of 30 action. It will be seen that SPLVs demonstrate these desirable charac:~eristics, while P~LVs do not.
There are two factors that cause vesicles to leak. One is auto-oxidation oî the lipids whereby the 35 hydrocarbon chains form peroxides which destabilize the 7'7 bilayer~. ~his oxidation can be dra~tically slowed down by the addition of an ioxidants 8UCh as butylated hydroxy toluene ~BHT) to the vesicle preparation. Ve~icles can aiso le~k becau~e agent6 i~ the exterior environment disrupt the bilayer organiza$ion of the lipid8 ~uch that ~he lipids remain intact, but the membrane develops a pore.
Pre~arations of lipid vesicles are white in color when first msde. Upon au~o-oxida~ion, ~he preparation 10 becomes discolored (browni~h). A comparison of MLVs to SPLVs prepared using the same lipid and aqueous components reveals tha~ MLVs discolos wi~hin one to two week~ whereas SPLY~ remain white for at least two months. This is supported by thin layer chromatography of the constituent 15 lipi~s which ~howed degradation of ~he lipids in ~he MLVs but not of the lipids of ~he 5PLV~. When ~hese ve~icles are prepared by adding BHT as well as the other constituents~ then MLVs appear slightly discolored within one month whereas the SPLVs remain white and appear stable 20 for at least 6 months and longer.
When placed in a bu~fer containing isotonic saline a~ neu~ral p~, SPLVs containing antibiotic are ~table for more than four months, as demonstrated in 25 Table X. These data indicate that none of the antibiotic originally encapsulated withln the SPLVs leaked out in the period of the experiment.
Other evidence indicates that SPLVs are able to 30 ~equester an encapsulated agent from molecules as ~mall as calcium ions for more than 5iX months~ Arsenazo III is a dye which changes color from red to blue with the slightest amount of divalent cation present. By encapsulating the dye in SPLVs and adding calcium chloride 35 to the stora~e buffer it is possible to measure the >77 -;L7-TABLE I
5STABILITY OF EGG P~QSPHATIDYLC~OLINE
SPLV~ A~l~ER STORAG~ IN SEALED
CONT~INERS AT ~C ~OR 4-1/2 ~ONTHS a Initial Læakage Bioavailabillty Entrapped ~:ntrapment ~nto of Entrapped Drug % Supernatantb Drug (%) 10 Strept~mycin Sulfate 34.1 0 97 Spectinomycin37.2 0 84 Chloramphenicol 35.~ 0 89 ~5 Oxytetracycline 1~.8 0 gl Erythromycin 0 D 4 0 97 Sulfamerazine 6 . 3 0 93 20 a 5PLVs were prepared using 127 ~M egg phosphatidylcholine (EPC~ and 25 IIM drug. At the end of 4 1/2 months storage at 4C the SPLVs were separated from storage buffex by centrifugation.
Ser ial dilutions of the SPLV contents and ~h supernatant were applied to bacterial lawns in order to determine bioactivity as compared to standard dilutions of an~ibiotic.
b o indicates below detectable levels stabili~y of the vesicles by looking for a color change.
The color remains undetectably different from its original rolor for at least 6.5 months, demonstrating that neither has the dye leaked out nor the ion leaked in~
These experiments demonstrate that SPLVs are sufficiently stable to withstand storage and handling -~.8-proklem60 ~l~hough i~ is possible ~o make MLVs which are ~ta~le ~or this long, they mus~: be made from synthetic lipids such as DSPC and thus become prohibitively expen~ive e 5.2.2. STA~ILITY O~ SPLVS IN OTHER ENVIRONMENTS
Placing lipid vesicles in a medium which contains membrane perturbing agents is a way to probe different 10 molecular organizations, Depending on how ~he membrane is organized, different ve~icles will respond differently to such agents~
In the following experiments vesicle~ were 15 prepared which contained a radioactive tracer molecule (3H inulin) within the o~cluded a~ueous compartment~
Inulin, a polysaccharide, partition into ~he aqueous phase, and thus when radiolabeled may be u~ed to tr~ce the aqueous contents of lipid vesicles. After an appropriate 20 interval of exposure to a given agent, the vesicles were separated from the loedium by centrifugation, and the relative amount of radi~activity that escaped from the vesicles into the medium was determined. These results are reported in Table II; valu~s are expre~sed as pexcent 25 leaked, meaning ~he proportion of radioactive material in the surrounding medium relative ~o the starting amo~nt encapsulated in 'che vesicles.
SPLV~ are more stable than MLVs in hydrochloric 30 acid. Table II illl3strates that both MLVs ~nd SPLVs, when ~de from egg lecithin, are destabili~ed when exposed to 0.125 N hydrochloric acid for one hour. i~owever, it is no~eworthy that the SPLVs are considerably less ~usceptible to ~he acid than MLVs~ Presumably this 3~ different response reflects an intrinsic difference in the way the lipids in~eract with their environment.
7~
TABLE I I
STA:3ILITY OF SPLVS IN OTEIER E:NVIPcONM;~3TS
Incubating Mediuma 96 LEAKAGE
MLVs SPLVs :~ydr och lor ic Ac id 0.125M 90.5 55.2 ~ Urea lM 21.7 44.8 Guanidine OoSM 5~7 7~
1.0M 303 10.1 15 Ammon i um Ac e ta te 0.5M 27.0 67~0 1~0M 25.9 54.7-63.1 Serum 76 . 2 57 . 8 20 a Incubation time is 2 to 4 hours except incubation in HCl was f or 1 hour .
SPLVs also re~pond different:Ly than MLVs when exposed to urea (FIG. 1 and Table II3. Urea is a molecule with both a chaotropic effect (disrupts the structure of water) and a strong dipole moment. It i~ observed that SPLVs are f~r more susceptible to urea than they are to an osmotic agent such as sodium chloride at the same 30 concentration (FIG. 1). MLV~ do not leak significantly more in urea than they would in ~odium chloride. Althou~h the explanations for this different behavior are ~heore~ieal, it would ~ppear that 'che response is due to ~he dipole effec~, rather than a chaotropic property9 35 ~ince guanidine, a molecule similar to urea, does not destabilize SPLVs (Table II). Although guanidine is also -;2~-~trongly chaotxopic, it doe~ no~ po~ses~ a ~rong dipole moment.
SPLVs are also su&ceptible to ammonium acetate, 5 while MLVs are not (Table II). ~owever, nei~her ~mmonium ion (in ammonium chloride~ nor acetate ~in ~odium acetate) ~re par~icularly effective in ~ausing SP~Vs ~o destabilize. ThuS it would appear ~hat i~ is no~ the ivn itself, but the polarity of the ammonium acetate which is 10 responsible for inducing leakage.
Initially these results ~eem urpri~ing because SPLVs are much more stable ~han MLVs ~hen incubated in body fluids such as ~era or blood. ~owever a ~heoretical 15 explanation for these re~ults can be proposed tof course other explanations ~re possible)7 If the stability of the SPLV is due to the unique structure of its membrane bilayers ~uch that the polar groups of the membrane lipids are hydrated by a cloud of oriented water molecules, or 20 hydration ~hell, then it i~ possible that any agent which disrupts or interferes with such hydration shells would promote changes in structural membrane integrity, and therefore, leakage.
Independent of the theoretical explanations for ~he destabilization of SPLVs in urea are correct, the results serve to demonstrate characteristic differences between the structure of MLVs and SPLVs. This difference serves a very useful purpose in application. As described 30 infra~ SPLVs become slowly leaky when applied ~o the eye.
Presumably this desired slow release of contents is due to a similar destabilization of the SPLVs when expose~ to tear fluid~
SPLVs are more stable in serum than MLVs. Many applications of lipid vesicles include administering them JrJ
intraperitoneally~ such as for the treat~ent of brucellosisO To be effec~ive, ~he vesicles mus~ survive for a ~u$ficien~ ~ime ~o reach their desired target~
SPLVs ~nd MLVs, both made from l~gg lecithin, were exposed 5 to fetal bovine erum which con~ained active complement, (Table II). After 48 hours exposure at 37C, ~PLV~ are demonstrably more ~table than MLVs.
5.2.3. CHARACIERISTICS O~ SPLVS ADMINISTERED I~ VIVO
SPLVs demonstrate a number of characteristics which make them particularly suitable as carriers for delivery systems in v~vo:
(A) SPLVs are resistant to clearance. When SPLVs are administered to an or~anism both the lipi~
component and the entrapped ac ive ingredient are retaine~
in the t~ssues and by the cells to which they sre administered;
~ B) SPLVs can be engineered to provide sustained selease. The stability of SPLVs i5 "adjustable" in that SPLVs are very stable during storage and are stable in the presence of body fluids but when administered ~.n vivo a 25 slow leakage of the active ingredient permits the sustained release of the ac~ive ingredient;
(C) Because of the high level of entrapment ana stability when administered, effective doses of the active 30 ingredient are rele~sed; and (D) The production of SPLVs is very cost effective in that stability o~ ~he vesicles is achieYed without incorpora~ing expensive stabilizers in~o ~he 35 bilayers.
6~ ~
~22-The following experimenks demonstrate ~ome of these characteris~ics of SPLVs when administered topically onto the eyes of test animal~. The SPLVs used in these experiments were prepared as previously ~e~rribed except 5 that the lipid bilayer and the active ingredie~t wer~ each radiolabeled in order to trace these component~ in the eye tissues over a period vf ~ime.
SPLVs were prepared using lOOmg egg 1C phosphatidylcholirJe (EPC) and lOOmg gentamycin sulfate.
The lipid component was radiolabeled by the incorporation of trace amo~nts of l25I-phosphatidylethanolamine (l25I-PE) into the bilayers, whereas ~he ac~ive ingredient in the aqueous phase was radiolabeled by ~he 15 addition of l25I-gentamycin sulfate (l25I~GS). The SPLVs were washed with buffer repeatedly in order to effectively remove unincorporated or unencapsulated materials~
Z An aliquot of the SPLV preparation was removed and extracted in order to separate the organic phase from the aq~eous phase. The radioactivity of each phase was measured in order to determine the initial ratio of I-PEo 5I-GS (cpm (counts per minute) in ~he lipid 25 phase:cpm in the aqueous phase) which was incorporated into the SPLVs.
The extraction was done as follows: 0.8ml of 0.4M NaCl (aqueous), l mQ chloroform, and 2 mQ methanol 30 were mixed to form a homogeneous phase. Then 4~l of the radiolabeled SP~Vs were added and mixed; as the SPLV
comp~nents dissolved into the organic phase and into the aqueous phase~ the mixture, which was initially ~urbid, became clear~ Th~ pha~es were -eparated b~ adding and 35 mixing lmQ 0.4M NaCl (aqueous~ and l m~ chloroform, which wa~ then centrifuged at 2,800 x 9 for 5 minute~, An aliquot (lmQ) s:)f ea~h phase was removed and the ~adioactivi'cy (in cpmj was mea~uredD (The initial ratio f 125I_pE 125I_~;S wa~ 1.55~
s ~ i~teen adult female SWi8S Webster mice were anesthetized and re~trained (in order to prevent them from wiping their eyes~O Equal aliquots t211~) of the radiolabeled SPLVs in ~uspension were topically applied to 10 each eye. Groups of three ~nimals were then acrificed at each of the following points: 1,, 2, 3, lB, and 24 hours.
Nine female Swiss Webs~cer mice (c:ontrols) were trea~ed identically except that equal aliquots (211Q) of an aqueous ~olution of radiolabeled gentamycin sulfate were applied 15 topically to ~ach eye. Groups of three control animals were ~acrificed at th~ end of 1, 4, and 8 hours.
Immediately ~fter sacrifice the eyelids of the animals were removed, minced, and extracted (using the 20 procedure previously described) in order to separate the aqueous components from the lipid components. The radioactivity of such phase was determined (as well as the ~otal number of radioactive counts recovered). The radioactivity measured in the lipid phase is an indication 25 of the retention of SPLV lipids by th~ eye tissue, whereas the radioactivity measured in the aqueous phase is an indication of the retention of gentamycin in the ey~
tissue. YIG. 2 graphically demonstrates the retention of each componen~ in the eyelid ~issue (expressed a~ ~he 30 percent of the original number of cpm applied to the eye).
~ IG~ 2 clearly demonstrates the retention of the SPLV lipid oomponent in the eyelid tissue over a 24 hour per iod, and the sustained release of gentamycin ~rom the 35 SPLVs ov r a 24 hour per iod (as reflected by the percen~c -2~-gentamy~in retained in the eyelid ti~sue ~uring his time). ~IGr ~ also demonstrates ~hat unencapsulated gentamycin taqueous gentamycin administered topically) is rapidly ~leared from th~ eyelicl ~issue. For example, 5 gentamycin in solution (control) was cleared from the eyelid tissue within 4 hours (less than 5~ of the gentamycin remained in the eyelid kis~ue~. ~n the other hand, more ~han 50% of the S~LV-encapsulated gentamycin was retained by the eyelid tissue in ~his 4 hour p~riod;
in fact, at the end of 24 hours more than 15% of the SPLV-encapsulated gentamycin was retained by the eyelid ~issue, This indicates that approximately 85~ of the SPLV-encapsulated gentamycin was released over a 24 hour period whereas 95% of the unencapsulated gentamycin 15 6ulfate wa5 ~leared within a 4 hour period~
~ able III compares the ratio of the SPLV lipid phase:aqueous phase retained in the eyelid tissue at each time point. An increase in this ratio indicates release 20 of gentamycin from the SPLVs.
The bioactivity of the SPLV-encapsulated gentamycin sulfate which was retained by the eyelid tissues was also evaluated, 5entamycin sulfate was 25 recovered from the eyelid tissues by removing an aliquot from the aqueous phase of the eyelid extracts prepared 3 hours after the SPLV-encapsulated gentamycin sulfate was applied to the eye. ~he a~ueous phase was serially diluted and 2~Q aliquots w~re placed onto S~aphylococcus 30 aureus lawns on agar plates; after 24 hours incubation the zones of inhibition were measured. The gentamycin sulfate recovered from the eyelid tissue extracts of animals treated with SPLV-encapsulated gentamycin ~ulfate fully retained its bioactivity.
6~
-:25~
~ABLE III
S~STAINED RELEASE O~ SPLV-ENCAPSVLATED
OENTAMYCIN AFTER ~OPICAL APPLICATION IN 2YES O~ ~ICE
Total SPLV Compo- Ratio of SPLV Lipid:
Time nent~ Recovered A~ueous Phase Post-Application from Eyelids Retained In Eyelids (~ Initial Dose) (1~5I-PE:~5I-5S) 1~ 0 10~% 1.55 lhr 100~ 2.1 3hr 100% 2.82 18hr 94% 6~89 24hr 85.1~ 7.17 5.2.4. ELECTRON SPIN RESONAMCE
Although SPLVs and MLVs appear identical by electron microscopy, ESR (electr~n spin resonance) spectroscopy reveals differences in their supramolec~lar Q~ructure. SPLVs can be distingui~hed ~rom MLVs on the basis of their molecular architecture as evidence by their increased molecular order J increased molecular motion and greater penetrability to ascorbate. It is likely that these differences in molecular architecture contribute to their àiffer2nt biological effects~
In electrorl spin resonanoe ~pectroscQpy a ~pin probe such as 5 doxyl stearate (5DS) is incorporated into the lipid bilayer. The unpaired electron of the doxyl gr oup absorb micr owave energy when the sample is inser ted 35 into a magnetic f aeld . The ~p~ctrum of the ab orption allows ~he determination of thre2 empiric~l parameters:
~:2h-S, the order p~rameter, ~O~ ~hla hyperfine couplin~
constant; and Tau ~he rotational correlation time. A
typical reading is ~hown in FIG. 3, wherein A i~ the SPLV
~ignal and B is the MLV signal, both are labeled with 5 5 ~oxy~ steara~e7 The spectra were taken a~ room temperature, scan range was 100 Gauss. The order parameter(s) which i~ dependen~ on both 2Tl and 2Tll measures the deviation of the observed ESR signal from the case of a completely uniform orientation of the probe.
1~ For a uniformly oriented sample S=l.0~, a random ~ample S=O. Th~ hyperfine coupling constant, Aol which can be calculated from 2Tl and 2Tll is considered to reflect local polarity and thus refl~cts the position of the spin probe within the ~embrane. The rotational correl~tion 15 time (which i~ dependent on WO' ho, h-l) can be thought of as the time required ~or the molecules to ~forget~ what their previous spatial orientatisns were, A
typical ESR de~ermination of the differences between SPLVs and MLVs having 5-DS as the 8pin probe is summarized in 20 Table IV.
Although in both cases the spin probe is reporting from the same depth in the bilayer, SPLVs possess a significantly greater degree of molecular order 25 and molecular motion than MLVs.
Another illustration of the differences between SPLVs and MLVs resides in the ability of ascorbate to reduce doxyl spin probes. It has been known fsr some time 30 that ascorbate reduces doxyl moieties presumably to their hydroxylamine analogs which do not ab~orb microwave energy in a magnetic f ield . In a~ueolls solutions the reduction occurs rapidly with concomitant loss of ESR signal. If the ~pin probe is in a protected environment 6uch as a 35 lipid bilayer it may be reduced more slowly or not at all TABLE IV
E.SR C~ARACTERI~ATION O~ SPLVS AND MLVS
Tau S Ao SPLVs 2965 x l0 9 Sec 0.614 14~9 10 MLVs 3.65 x l0 9 Sec On595 14~g by the hydrophilic ascorbate. Thus the rate of nitroxide reduction can be used to ~tudy the rate of penetra ion of 15 the ascorbate into lipid bilayer~. FI~c 3 ~hows the percentage remaining ~pin versus time or SPLVs and ~LVs ~u~pended in an ascorbate ~olution. At 90 minutes the ascorbate has reduced 25% of the probe embedded in MLVs ~ut 60~ of the probe embedded in 5PLVs~ SPLVs allow for a 20 drama~ioally greater pene~rabil~ty of ascorbate than do MLVs.
5 5 ~ ~ 5~ ENTRAPMENT OF ACTIVE MATERIAL BY SPLVs As another prime ~xample of the superiority of SPLVs over traditional ~LVs, SPLVs entrap a larger percentage of the available ac~ive material thereby con~erving material (see Table V).
5.~.6~ INTERACTION O~ SPLVS WITH CELLS
Still another benefit of SPLVs is that SPLVs interact with ~ells such that a relatiYely large portion of the ma~erials encapsulated inside ~he vesicle is 35 dispersed throughout the cytoplasm of the rell~ rather l~Le~86 ~7 ~ABLE V
COMPAP~ISC~N OF MLVS AND SPLVS
9~ Available ~5a~erial Entrapped MLVs SPLVs Encapsulation of:
inulin (aqueous 2 6% 20-30 space mar ker ) bovine serum 15% 20-50 albumin streptomyci~ 12-1596 20-40~6 polyvinylpyrrolidone 5~ 25-35%
(aqueous space) than being limited to phagocytic vesicle~. When SPLVs are mixed wi th cells the two appear to coale~ce . By coalescence, SPLVs, unlike MLVs, interact with cells in vitro so that all the cells contain at least some of the materials originally entrapped in the SPLVs. This ma~erial appears to be distributed throughout each cell ~nd no~ limi~ed to just the phagocytic vesicles. This can be demonstrated by incorporating ferritin in the aqueous phase of a SPLV preparation. After coalescence with a cell in culture, ultras'cructural analysis reveals ~hat ~he ferrit.irl is distribu~ed throughout ~he cytosol and is not bound by in~racellular membranes. While ~hi~ phenomenon can be shown to occur with PqLVs a greater quantity of material can be transferred by SPLVs.
~22-The following experimenks demonstrate ~ome of these characteris~ics of SPLVs when administered topically onto the eyes of test animal~. The SPLVs used in these experiments were prepared as previously ~e~rribed except 5 that the lipid bilayer and the active ingredie~t wer~ each radiolabeled in order to trace these component~ in the eye tissues over a period vf ~ime.
SPLVs were prepared using lOOmg egg 1C phosphatidylcholirJe (EPC) and lOOmg gentamycin sulfate.
The lipid component was radiolabeled by the incorporation of trace amo~nts of l25I-phosphatidylethanolamine (l25I-PE) into the bilayers, whereas ~he ac~ive ingredient in the aqueous phase was radiolabeled by ~he 15 addition of l25I-gentamycin sulfate (l25I~GS). The SPLVs were washed with buffer repeatedly in order to effectively remove unincorporated or unencapsulated materials~
Z An aliquot of the SPLV preparation was removed and extracted in order to separate the organic phase from the aq~eous phase. The radioactivity of each phase was measured in order to determine the initial ratio of I-PEo 5I-GS (cpm (counts per minute) in ~he lipid 25 phase:cpm in the aqueous phase) which was incorporated into the SPLVs.
The extraction was done as follows: 0.8ml of 0.4M NaCl (aqueous), l mQ chloroform, and 2 mQ methanol 30 were mixed to form a homogeneous phase. Then 4~l of the radiolabeled SP~Vs were added and mixed; as the SPLV
comp~nents dissolved into the organic phase and into the aqueous phase~ the mixture, which was initially ~urbid, became clear~ Th~ pha~es were -eparated b~ adding and 35 mixing lmQ 0.4M NaCl (aqueous~ and l m~ chloroform, which wa~ then centrifuged at 2,800 x 9 for 5 minute~, An aliquot (lmQ) s:)f ea~h phase was removed and the ~adioactivi'cy (in cpmj was mea~uredD (The initial ratio f 125I_pE 125I_~;S wa~ 1.55~
s ~ i~teen adult female SWi8S Webster mice were anesthetized and re~trained (in order to prevent them from wiping their eyes~O Equal aliquots t211~) of the radiolabeled SPLVs in ~uspension were topically applied to 10 each eye. Groups of three ~nimals were then acrificed at each of the following points: 1,, 2, 3, lB, and 24 hours.
Nine female Swiss Webs~cer mice (c:ontrols) were trea~ed identically except that equal aliquots (211Q) of an aqueous ~olution of radiolabeled gentamycin sulfate were applied 15 topically to ~ach eye. Groups of three control animals were ~acrificed at th~ end of 1, 4, and 8 hours.
Immediately ~fter sacrifice the eyelids of the animals were removed, minced, and extracted (using the 20 procedure previously described) in order to separate the aqueous components from the lipid components. The radioactivity of such phase was determined (as well as the ~otal number of radioactive counts recovered). The radioactivity measured in the lipid phase is an indication 25 of the retention of SPLV lipids by th~ eye tissue, whereas the radioactivity measured in the aqueous phase is an indication of the retention of gentamycin in the ey~
tissue. YIG. 2 graphically demonstrates the retention of each componen~ in the eyelid ~issue (expressed a~ ~he 30 percent of the original number of cpm applied to the eye).
~ IG~ 2 clearly demonstrates the retention of the SPLV lipid oomponent in the eyelid tissue over a 24 hour per iod, and the sustained release of gentamycin ~rom the 35 SPLVs ov r a 24 hour per iod (as reflected by the percen~c -2~-gentamy~in retained in the eyelid ti~sue ~uring his time). ~IGr ~ also demonstrates ~hat unencapsulated gentamycin taqueous gentamycin administered topically) is rapidly ~leared from th~ eyelicl ~issue. For example, 5 gentamycin in solution (control) was cleared from the eyelid tissue within 4 hours (less than 5~ of the gentamycin remained in the eyelid kis~ue~. ~n the other hand, more ~han 50% of the S~LV-encapsulated gentamycin was retained by the eyelid tissue in ~his 4 hour p~riod;
in fact, at the end of 24 hours more than 15% of the SPLV-encapsulated gentamycin was retained by the eyelid ~issue, This indicates that approximately 85~ of the SPLV-encapsulated gentamycin was released over a 24 hour period whereas 95% of the unencapsulated gentamycin 15 6ulfate wa5 ~leared within a 4 hour period~
~ able III compares the ratio of the SPLV lipid phase:aqueous phase retained in the eyelid tissue at each time point. An increase in this ratio indicates release 20 of gentamycin from the SPLVs.
The bioactivity of the SPLV-encapsulated gentamycin sulfate which was retained by the eyelid tissues was also evaluated, 5entamycin sulfate was 25 recovered from the eyelid tissues by removing an aliquot from the aqueous phase of the eyelid extracts prepared 3 hours after the SPLV-encapsulated gentamycin sulfate was applied to the eye. ~he a~ueous phase was serially diluted and 2~Q aliquots w~re placed onto S~aphylococcus 30 aureus lawns on agar plates; after 24 hours incubation the zones of inhibition were measured. The gentamycin sulfate recovered from the eyelid tissue extracts of animals treated with SPLV-encapsulated gentamycin ~ulfate fully retained its bioactivity.
6~
-:25~
~ABLE III
S~STAINED RELEASE O~ SPLV-ENCAPSVLATED
OENTAMYCIN AFTER ~OPICAL APPLICATION IN 2YES O~ ~ICE
Total SPLV Compo- Ratio of SPLV Lipid:
Time nent~ Recovered A~ueous Phase Post-Application from Eyelids Retained In Eyelids (~ Initial Dose) (1~5I-PE:~5I-5S) 1~ 0 10~% 1.55 lhr 100~ 2.1 3hr 100% 2.82 18hr 94% 6~89 24hr 85.1~ 7.17 5.2.4. ELECTRON SPIN RESONAMCE
Although SPLVs and MLVs appear identical by electron microscopy, ESR (electr~n spin resonance) spectroscopy reveals differences in their supramolec~lar Q~ructure. SPLVs can be distingui~hed ~rom MLVs on the basis of their molecular architecture as evidence by their increased molecular order J increased molecular motion and greater penetrability to ascorbate. It is likely that these differences in molecular architecture contribute to their àiffer2nt biological effects~
In electrorl spin resonanoe ~pectroscQpy a ~pin probe such as 5 doxyl stearate (5DS) is incorporated into the lipid bilayer. The unpaired electron of the doxyl gr oup absorb micr owave energy when the sample is inser ted 35 into a magnetic f aeld . The ~p~ctrum of the ab orption allows ~he determination of thre2 empiric~l parameters:
~:2h-S, the order p~rameter, ~O~ ~hla hyperfine couplin~
constant; and Tau ~he rotational correlation time. A
typical reading is ~hown in FIG. 3, wherein A i~ the SPLV
~ignal and B is the MLV signal, both are labeled with 5 5 ~oxy~ steara~e7 The spectra were taken a~ room temperature, scan range was 100 Gauss. The order parameter(s) which i~ dependen~ on both 2Tl and 2Tll measures the deviation of the observed ESR signal from the case of a completely uniform orientation of the probe.
1~ For a uniformly oriented sample S=l.0~, a random ~ample S=O. Th~ hyperfine coupling constant, Aol which can be calculated from 2Tl and 2Tll is considered to reflect local polarity and thus refl~cts the position of the spin probe within the ~embrane. The rotational correl~tion 15 time (which i~ dependent on WO' ho, h-l) can be thought of as the time required ~or the molecules to ~forget~ what their previous spatial orientatisns were, A
typical ESR de~ermination of the differences between SPLVs and MLVs having 5-DS as the 8pin probe is summarized in 20 Table IV.
Although in both cases the spin probe is reporting from the same depth in the bilayer, SPLVs possess a significantly greater degree of molecular order 25 and molecular motion than MLVs.
Another illustration of the differences between SPLVs and MLVs resides in the ability of ascorbate to reduce doxyl spin probes. It has been known fsr some time 30 that ascorbate reduces doxyl moieties presumably to their hydroxylamine analogs which do not ab~orb microwave energy in a magnetic f ield . In a~ueolls solutions the reduction occurs rapidly with concomitant loss of ESR signal. If the ~pin probe is in a protected environment 6uch as a 35 lipid bilayer it may be reduced more slowly or not at all TABLE IV
E.SR C~ARACTERI~ATION O~ SPLVS AND MLVS
Tau S Ao SPLVs 2965 x l0 9 Sec 0.614 14~9 10 MLVs 3.65 x l0 9 Sec On595 14~g by the hydrophilic ascorbate. Thus the rate of nitroxide reduction can be used to ~tudy the rate of penetra ion of 15 the ascorbate into lipid bilayer~. FI~c 3 ~hows the percentage remaining ~pin versus time or SPLVs and ~LVs ~u~pended in an ascorbate ~olution. At 90 minutes the ascorbate has reduced 25% of the probe embedded in MLVs ~ut 60~ of the probe embedded in 5PLVs~ SPLVs allow for a 20 drama~ioally greater pene~rabil~ty of ascorbate than do MLVs.
5 5 ~ ~ 5~ ENTRAPMENT OF ACTIVE MATERIAL BY SPLVs As another prime ~xample of the superiority of SPLVs over traditional ~LVs, SPLVs entrap a larger percentage of the available ac~ive material thereby con~erving material (see Table V).
5.~.6~ INTERACTION O~ SPLVS WITH CELLS
Still another benefit of SPLVs is that SPLVs interact with ~ells such that a relatiYely large portion of the ma~erials encapsulated inside ~he vesicle is 35 dispersed throughout the cytoplasm of the rell~ rather l~Le~86 ~7 ~ABLE V
COMPAP~ISC~N OF MLVS AND SPLVS
9~ Available ~5a~erial Entrapped MLVs SPLVs Encapsulation of:
inulin (aqueous 2 6% 20-30 space mar ker ) bovine serum 15% 20-50 albumin streptomyci~ 12-1596 20-40~6 polyvinylpyrrolidone 5~ 25-35%
(aqueous space) than being limited to phagocytic vesicle~. When SPLVs are mixed wi th cells the two appear to coale~ce . By coalescence, SPLVs, unlike MLVs, interact with cells in vitro so that all the cells contain at least some of the materials originally entrapped in the SPLVs. This ma~erial appears to be distributed throughout each cell ~nd no~ limi~ed to just the phagocytic vesicles. This can be demonstrated by incorporating ferritin in the aqueous phase of a SPLV preparation. After coalescence with a cell in culture, ultras'cructural analysis reveals ~hat ~he ferrit.irl is distribu~ed throughout ~he cytosol and is not bound by in~racellular membranes. While ~hi~ phenomenon can be shown to occur with PqLVs a greater quantity of material can be transferred by SPLVs.
7~7 5 . 2 ., '1. BtlOYANT DE~ SITY O~ SPI,VS
Additionally9 SPLVs have a lower buoyant density than P~LVs. This is mea~ured by banding in ~ ~icol 5 gradient (see Table VI)~, 5 . 2 . 8 . VOLUME OF SPL~IS
Fur thermore, when collec:ted in a pellet by 1~ centrifugatiorl from 1"~00 to 100,000 x 99 SPLVs form a pellet that i~ substantially larger than ML~s, given the s~me phospholipid concentration. At a force of 16, 000 x 9, the SPLVs form ~ pellet approximately one third larger th an MLVs .
5 r 2 . 9 . (: ~M O~ I C PR OP E Rq I E S O~ S P LV S
Since phospholipid bilayers are permeable to water, placirlg MLVs in a hypertonic environr~ent drives the 20 c~c:cluded water out due to osmotic force. SPLVs shrink more than MLVs. In addition, afte~ shrinking 16 hours in a buf fer that is twenty times higher ~han the in~ernal salt concentration, SPLV~ do not shrink to the same final volume as MLVs (SPLV pellets remain 1/3 l~rger than E9LV
25 pellets), ~his indical~e~ that the difference in pellet size is not due to differerlces in aqueous enclosed volumeO
5 . 3 ~ t~SES O~ SPLVS
SPLVs are particularly u&eful in ~ystems where the following factors are important: s~ability during stora~e and con'cact wilth budy fluids; a relatively high degree of encapsulation; cos~-effectiverless; and the release of the entrapped compound in it~ biologie:ally 35 ~c t ive f orm .
TABLE VI
BUOYANT DEM SITY
MLVs PLV~
in ficol layers above 19~ layers above 0.5%
in gms/mQ 1.û71 1.0274 ~ urthermore, depending upon the mode of administration in vivo, SPI.Vs can be resi ~ant to rapid clearance (~, where sus~ained delivery i~ important) or 15 can be delivered to ~he cells of ~he RES.
As a result, the SPLVs of the invention are usefully employed in a wide variety of systems. They may be used to enhance the therapeutic efficacy of 20 me~ications, to cure infections, to enhance enzyme replacement, oral drug delivery, topical drug delivery, for introducing gene'cic information into cells in vitro and in vivo, for the production of vaccines, for the introduction of recombinant deoxyribonucleic acid segments 25 into cells, or as diagnostic reagen~s for clinical tests following release of entrapped ~reporter" molecules. The SPLVs can also be employed to encapsulate cosrnetic preparations/ pesticides, compounds for sustained slow release to effect the growth of plant~ and the like.
The methods which follow~ while described in terms of the use of SPLVs, cs:~ntemplate the use of SPLVs or any c~ther liposome or lipid vesicle having furlctl~nal charac~eristics similar to those of SPLVs~
5~3~lo DELX~ERY 0~ BIOACTIVE COMPOUNDS
Delivery of compou~ds to cells ln itro (~
animal cells, plant cells, protifits~ et ~ generally 5 requires the addi~ion o~ the SPLVs containing th~ compound to the cell~ in culture. SPLYs, howe~er~ can ~l~o be used to deliver compounds an ivo in animals ~including man), plants and protists. Depending upon the purpo5e of delivery, the SPLVs may be administered by a number of 10 routes: in man and animal~ this includes but is not limited to injection (~ , intravenous, intraperitoneal, intramuscular, ~ubcutaneous, intraauricular, intr~ra~ry, intraureth ally~ etc~), topical application (~ on afflicted areas), and by absorption through epithelial or 15 mucocutaneous linings (e.~., ocular epithelia, oral mucosa, rectal an~ ~aginal epithelial lining~, the respiratory tract linings, nasopharyngeal mucosa, intestinal m~cosa, etc.); in plants and protist~ this includes but is not limi~ed ~o direct applica~ion to 20 organism, dispersion in the organism~s habitat, addition to the surrounding environment or surrounding water, etc.
The mode of application may also determine ~he sites and cells in the organism to which th compound will 25 be delivered. For in tance, delivery to a specific si~e of infection may be most easily accomplished by topical application (if the infection is external). Delivery to the cir~ulatory system ~and hence reticuloendothelial cells), may be most easily accomplished by intravenous, 30 intr~peritoneal, intramuscular, or ~ubcutaneous injections.
Since SPLVs allow for ~ sustained release of the compound, doses which may otherwise be toxic to the organism may be utilized in one or more admini~trations to : 35 the organism.
3 Ei77 The ~ections which fol.low de~cr~be ome overall ~chemes in which SPLVS may be u~ed and demons~rate but do no~ limi~ the scope of the pre~;ent invention.
5.302. TREATMENT OF PATHOLOGIES
A number of pathological conditions which OCGUr in man, animal~ and plant~ may be ~reated more effectively by encapsulatiny the appropriate compound or compounds in 10 SPLVs. These pa~hologic condi~ion~ in~lude bu~ are not limited to infections (intracellular ~nd extracellular);
cysts, tumors and tumor cells, allergies~ etc.
~any strategies are possible for using SPLVS in 15 the treatmen~ of such pa~hologies; a few overall ~chemes are outlined below which are particulazly usef~l in that they take advantage of the faot that SPLVs when administered in vivo are internalized by macrophages.
~n one scheme, SPLVs are used to deliver therapeutic agents to sites of intracellular infections.
Certain diseases inYolve an infection of cells of the reticuloendothelical system, e~, brucellosisO These intracellular infections are difficult to cure for a 25 number of reasons: ~l) because the infectious organisms reside within the cells of the reticuloendothelial system, they are sequestered from circulating therapeutic agents which c~nnot cross the cell membrane in therapeutically ~uf~ic~ent concentrations, and, there~ore, are highly 30 resistant to treatment; (2) often the administration of toxic levels of therapeutic agents are required in order to combat such infections; and (3) the treatment has to be ~ompletely effeotive because any residual infection after treatment can reinfect the host organism or can be 35 tran~mitted to sther hosts.
~.i3-According to one m~de of the pre~ent invention, SPLVs containing an appropriate biologically active compound are adminis~ere~ (pre:Eer~bly intr~peritoneally or intravenously) to the ho~t organism or potQntial host 5 or~anism (e.~., in animal herds, ~he uninf~cted animals as wPll as infected animals may be treated)O 5ince phagvcytic cells internalize SPLVs, the admini~tration of an SPLV-encapsulated substance that is biologically active a~ainst the infecting organism will result in directing the bioactive substance to the site o~ infection. Thus, the method o~ the present invention may be used to eliminate infection caused by a variety of microorganisms~
bacteria, parasites, fungi, mycoplasmas, and viruses, including but not limited to: Brucella spp., 15 Mycobacterium ~p~, Salmonella sEp., Listeria pp., F~anci~ella ~ , Hi~topla~ma sp~, Corynebacteri~m spp., Coccidiodes ~e~ and lymphocytic choriomeningitis virus.
The therapeutic agent selected will depend upon 20 the organism causing the infection. ~or instance, bacterial infections m~y be eliminated by encapsulating an antibiotic. The antibiotic can be contained within the aqueous fluid of the 5PLV andJor inserted into the vPsicle bilayer. Suitable antibiotics include but are not limited to: penicillin, ~mpicillin, hetacillin, carbencillin, tetracycline, tetracycline hydrochloride, oxytetracycline hydrochloride, chlortetracycline hydrochloride, 7-chloro-6-dimethyltetracycline, doxycycline monohydrate, methacycline hydrochloride, minocycline hydrochloride, 30 rolitetracycline, dihydrostreptomycin, streptomycin, gentamicin, kanamycin, neomycin, erythromycin, carbomycin, oleandomycin, troleandomycin, Polymyxin B collistin, cephalothin sodium, cephaloridine, cephaloglycin dihydrate, and cephalexin monohydrate.
-.3~-~ e have demon~tra~ed the effec~iveness of ~uch treatment~ in curing brucellosis (~ee Examples, infra).
By ~he procedure of ~his invention~ the effectiveness and duration of action are pro~ong,ed. It i8 surprising that 5 ~his sy~tem i~ effec~ive for treating infe tion~ which do not respond to known treatments suc~ a~ antibiotics entrapped in MLVs~ 5uccessful trea~ment is unexp~cted since any small remaining in~ections will spread and the infectious cycle will commence again. WP have also 10 demons~rated success in ~reating lymphocy~ic choriomeningitis virus infection.
Of oourse, the invention is not limited to trea~men~c of in~cracellular infections. The SPLVs can be 15 ~irected to a~varie~y of sites of infection whether intracellular or extracellular. For inhtance, in ano~her embodiment o~ the prese~t invention, macrophages ~re used to carry an active ~gent to the site of a systemic extracellular infection~ According to this scheme, SPLVs 20 are used to deliver ~ therapeutic subs~ance to uninfected macrophages by administering the SPLVs in vivo (preferably intraperitoneally or intravenously). The macrophages will coalesce with the SPLVs and then become "loaded't with the therapeutic substance; in general, the macrophages will 25 retain the substance for approximately 3 to S days. Once the ~loaded" macrophagec reach the site of infection, the pathogen will be internalized by the macrophages. As a resultJ the pathogen will contact the therapeutic substance contained within the macrophage, and be 30 destroyed~ This embodiment of $he invention is particularly useful in the treatment of Staphylococcus aureus mas~itis in man and cattle.
If the site of infection or a~fliction i5 35 external or ~csessible the SPLV-entrapp~d SherapeuSic _31j_ ~gent can be ~pplied top~cally. ~ particularly u e~ul application involves the treatment of eye afflictions. In the case of ocular a~lictio~ls, SPLVs containing one or more appropria~e active ~ngredien~s may be applied 5 topically to the afflicted eye. A number of organisms cause eye infections in animals and man. Such organisms include but are not limited to: ~oraxell~
Clostridium pp., Corynebacterium ~ , Diplococcus ~p., ~lavobacterium spp~, ~emophilus ~ , Rlebsiella spp., 10 Leptospira ~ r ~ycobacterium ~pp., Nei~seria Propionibacterium ~, Proteus ~ , Pesudomonas ~
Serratia ~ , Escherichia ~ , Staphyloco cus sp~., St~ept~coccus ~eP~ and bacteria-like organisms including Mycoplasma ~ nd Rickettsia ~ These in~ections are 15 difficult ~o eliminate using conven~ional methods because any residual infection remaining after treatmen~ can reinfect through lacrimal ~ecretions. We have demonstrated the use of SPLVs in curing ocular infections caused by Moraxella bovis (see examples, infra).
Because 5PLVs are resistant to clearance and are capable of sustained release of their conten~s, SPLVs are als3 useful in the ~reatment of any affliction requiring prolonged contact with the active treating substance. ~or 25 example, glaucoma is a disorder characterized by a gradual rise in intraocular pressure causing progressive loss of peripheral vision, and, when uncontrolled, loss of central vision and ultimate blindness. Drugs used in the treatment of glaucoma may be applied topically as 30 eyedrops, However, in some cases treatment requires administering drops every 15 minutes due to the rapid cleariny of the drug from the eye socket~ If an affliction such as glaucoma is to be treated by this invention ~herapeutic substances as pilocarpine, 35 ~loropryl, physostigmine, carcholin, aceta~olamide, 6~7 .
ethozol~ide, dichlorphenamide~ carbachol, demecarium bromide~ diisopropylphosphofluoridate t ecothioplate iodide, physostigmine, or neo~tigmine, etc7 can be entrapped within SPLVs which are then applied to the 5 ~ffected eyeO
Other agent~ which may be encapsulated in SPLVs and applied topically include but are not limited to:
mydriatics ~e.~., epinephrine, phenylepinephrineg hydroxy 10 amphetamine, ephedrine, atropine, homatropine, ~copolamine, cyclopentolate, ~ropicamide, encatropine, etc.); local anesthetics; antiviral agent~
~doxuridine, adenine arabinoside, etc.); antimycotic agents (e~y~, amphoteracin B, na amycin, pimaricin, 15 ~lucyto~ine, nystantin, thimerosal, ~ulfamerazine, thiobendazole, tolnh f~ate, ~risiofulvin, etc.);
antiparasitic agents (~ sulfonamide~, pyrimethamine, clindamycin, etc.); and anti-inflammatory agents (e.g., corticosteriods such as ACTH, hydrocortisone, prednisone, 20 medrysone, beta methasone, dexamethasone, fluoromethalone, triamcinalone, etc.).
The following Examples are given for purposes of illustration and not by way of limitation on the ~cope of 25 the inverltion.
6. EXAMPLE: PREPP.RA~ION OF SPLVS
As explained in Section ~.l. the basic method for 30 preparing SPLVs involves dissolviny a lipid or mixture of lipids into an organic solvent, adding an aqueous phase and the mater ial to be ~ncapsulated, and ~onicating the mixture. In the preferred embodiment the solvent is removed during sonication; however, the organic solvent 35 may be removed dur ing or af ter sonication by any ~37-evapora~ive technique. The S~I.Vs u~ed in ~11 of ~he examples contained herein were prepared as described in the following su~sections (however any method which yields SPLVs may be used)~
6.1~ SPLVS CONTAINING ANTIBIOTICS
A 5 mQ diethyl ether solution of lO0 mg leci thin was prepared. The mixture was pla¢ed in a round-bottom 10 flask O Then a solution (0. 3 mQ) containing lO0 mg of s~reptomycin ~ulfate at pH 7 .. d~ in 5 ~ ~IEP~:S
(4- [2 Elydroxyethyl] piperazino 2-ethane sulfonic acid~/0.0725 P~ NaCl/0.0725 M KCl was pipetted into the glass vessel ~ontaining ~he diethyl ether ~olution of 15 lipid. The mixture was placç~d in a bath ~onicator (Laboratory Supplie~ Col. ~ Inc. ) type 10536 for several minutes, (80 kHz frequency:ou~put 80 watts~ while being dried to a viscous paste by passing thereover a gentle stream of nitrogen.
To the viscous paste remaining was added 10 m~ of 5 mM HEPES. The resulting SPLV preparation, containing streptomycin, was suspended in the buffer solution, shaken for ~everal minutes on a vortex mixer, and freed of 25 ~on-encapsulated streptomycin by centrifuging at 12,000 x g for 10 minutes at 20~C. The resulting cake was suspended in 0.5 mQ of 5 mM HEPES.
The procedure described above was followed except 30 that strep~omycin was substituted by each one of the following. dihydrostreptomycin, gentamycin sulfate, ampicillin, tetracyline hydrochloride, and kanamycinO
6~
-3~-6,20 SPLVS CO~TAINING CTHER ~EMBR~NE C~NSTI~UENTS
The proces~ de~cribed in Section 6.1~ was followed ~xcept that any one of ~he following w~s added 5 with ~he egg lecithino (1) phosphatidic acid to give a molar ratio or 8:2 ~lecithin:di~etylphosphate~; (2) stearylamine to give a ~olar r~tio of 8:2 ~lecithin:
~tearylamine); cholest~rol and stearylamine to give a molar ratio of 7:2:1 (lecithin:cholesterol:~tearylamine);
10 and (3) phosphati~ic acid and chole~terol to give a molar ratio of 7:2:1 (lecithin:phosphatidic acid:choles~erol).
6.3. SPLVS CONTAINING PILOCARPINE
The procedure of Section 6cl. wa~ followed except that the antibioti~ ~treptomy~in was replaced with pilocarpine .
~4s SPLVS P~EPARED ~ITH AND WITHOUT BHT
IJndistilled ether contain~ an anti-oxidant, 1 ~g/mQ butylhydroxytoluene (BHT), ~or storage purposes.
The procedure described in Section 6.1. was following using undistilled ether as the solvent in order to 25 incorporate BE~T into the SPLV preparation.
In order to prepare SPLVs without incorporation of BMT, the procedur~ described in Section 6.1. was followed using distilled ether as the solventO
7. EXAMPLE: SPLV MEDIATED DELIVERY IN VITRO
In the following exampl , SPLV mediated delivery of antibiotics to macrophages in culture was demonstrated.
6'7~7 -3~
Peritoneal n~acrc~phage~; were obt~ ed by per itoneal lavage from C5733L~ adult male mice and su~pended in minimal essential medillm (MloE~ pEI 7 1> 2 containing 1096 heat-inactivate~ fetal calf ~erum. Cells 5 were ~u~pended at a concentra~ion oiE 1 ~ 106 cell~ per mQ in 96 well tis~ue s:ul~ure di~hes. To culture~
containing adherent peritoneal macrophages, were added B.
canis at concentraticns of 1 x 106 OEU (colony forming uni~) per m~. After 12 ho~rs, bac~eria no~ engulfed by 10 peritoneal macrophages were removed by repeated washings with ~.E.M. Af er washing of peri~oneal macrophage cul~ures, ~hey were divided into 5 groups, each con~aining 12 replicate cul~ures per group~ ~roup 1, designa~ed Controls~ received no treatmentO Group 2 received aqueous 15 ~treptomycin ~ulfate at a concentration of 1 mg~m~. Group 3 received buffer-filled SPLV~. Group 4 received aqueous ~treptomy~in ulfate (1 mg/mQ) plu5 preformed buffer-filled SPLVs. Group 5 received SPLVs containing treptomycin sulfate (1 mg/mQ)~ After 24 hours, super-natants were removed by repeated washings and peritoneal macrophages were di~rupted by repeated free~ing and thawing. ~erial dilutions of disrup~ed macrophages were plated onto brucella agar and, after 4 days, surviving B.
canis were determined by limiting dilution techniques.
25 Results shown in Table VII indicate that SPLV-entrapped ~treptomycin was totally efective in killing and eliminating the intracellular B. canis infec~ion in vitro.
~he experiment was repeated with Bo abortus 3D exactly as described above except that peritoneal macrophages were obtained by peritoneal lavage from adult female albino guinea pigs. Results are also ~hown in Table ~.1~ I .
TA~LE VII
COLONY-~ORMING ~ S OF INTRA OE LL~LAR
RRUCELLA ISOLAI'ED ~q`~R ~REA~MEN~ OF IN~ECTED
~ACROPHAGES WITH SPLVS CONTAINING STREPTOMYCI~
B~ c~ni~a B. abortusb Controls2.6~1~13x103 3.1+~81x104 Buffer filled~.B2~0.10x1032.9+0.17x104 SPLVs Free 3.11+0.40x103 3.3~0~25x104 StreptomycinC
Streptomycin2.76~0.20x1032.8+0.42x104 15 Plu~ Buffer- .
filled SPLV~C
SP1V-Entrapped 0 0 StreptomycinC
20 a Colony forming units ~CFU) of B. canis ~mean + SD of 12 replicates) i~olated from equal numbers of previously infected mouse (C57Blk) peritoneal macrophages.
b C~U of B. abortus ~mean ~ SD of 12 replicates) isolated from equal numbers of previously infected guinea pi~ peritoneal macrophages.
Concentration of streptomycin 1 mg/mQ.
Additionally9 SPLVs have a lower buoyant density than P~LVs. This is mea~ured by banding in ~ ~icol 5 gradient (see Table VI)~, 5 . 2 . 8 . VOLUME OF SPL~IS
Fur thermore, when collec:ted in a pellet by 1~ centrifugatiorl from 1"~00 to 100,000 x 99 SPLVs form a pellet that i~ substantially larger than ML~s, given the s~me phospholipid concentration. At a force of 16, 000 x 9, the SPLVs form ~ pellet approximately one third larger th an MLVs .
5 r 2 . 9 . (: ~M O~ I C PR OP E Rq I E S O~ S P LV S
Since phospholipid bilayers are permeable to water, placirlg MLVs in a hypertonic environr~ent drives the 20 c~c:cluded water out due to osmotic force. SPLVs shrink more than MLVs. In addition, afte~ shrinking 16 hours in a buf fer that is twenty times higher ~han the in~ernal salt concentration, SPLV~ do not shrink to the same final volume as MLVs (SPLV pellets remain 1/3 l~rger than E9LV
25 pellets), ~his indical~e~ that the difference in pellet size is not due to differerlces in aqueous enclosed volumeO
5 . 3 ~ t~SES O~ SPLVS
SPLVs are particularly u&eful in ~ystems where the following factors are important: s~ability during stora~e and con'cact wilth budy fluids; a relatively high degree of encapsulation; cos~-effectiverless; and the release of the entrapped compound in it~ biologie:ally 35 ~c t ive f orm .
TABLE VI
BUOYANT DEM SITY
MLVs PLV~
in ficol layers above 19~ layers above 0.5%
in gms/mQ 1.û71 1.0274 ~ urthermore, depending upon the mode of administration in vivo, SPI.Vs can be resi ~ant to rapid clearance (~, where sus~ained delivery i~ important) or 15 can be delivered to ~he cells of ~he RES.
As a result, the SPLVs of the invention are usefully employed in a wide variety of systems. They may be used to enhance the therapeutic efficacy of 20 me~ications, to cure infections, to enhance enzyme replacement, oral drug delivery, topical drug delivery, for introducing gene'cic information into cells in vitro and in vivo, for the production of vaccines, for the introduction of recombinant deoxyribonucleic acid segments 25 into cells, or as diagnostic reagen~s for clinical tests following release of entrapped ~reporter" molecules. The SPLVs can also be employed to encapsulate cosrnetic preparations/ pesticides, compounds for sustained slow release to effect the growth of plant~ and the like.
The methods which follow~ while described in terms of the use of SPLVs, cs:~ntemplate the use of SPLVs or any c~ther liposome or lipid vesicle having furlctl~nal charac~eristics similar to those of SPLVs~
5~3~lo DELX~ERY 0~ BIOACTIVE COMPOUNDS
Delivery of compou~ds to cells ln itro (~
animal cells, plant cells, protifits~ et ~ generally 5 requires the addi~ion o~ the SPLVs containing th~ compound to the cell~ in culture. SPLYs, howe~er~ can ~l~o be used to deliver compounds an ivo in animals ~including man), plants and protists. Depending upon the purpo5e of delivery, the SPLVs may be administered by a number of 10 routes: in man and animal~ this includes but is not limited to injection (~ , intravenous, intraperitoneal, intramuscular, ~ubcutaneous, intraauricular, intr~ra~ry, intraureth ally~ etc~), topical application (~ on afflicted areas), and by absorption through epithelial or 15 mucocutaneous linings (e.~., ocular epithelia, oral mucosa, rectal an~ ~aginal epithelial lining~, the respiratory tract linings, nasopharyngeal mucosa, intestinal m~cosa, etc.); in plants and protist~ this includes but is not limi~ed ~o direct applica~ion to 20 organism, dispersion in the organism~s habitat, addition to the surrounding environment or surrounding water, etc.
The mode of application may also determine ~he sites and cells in the organism to which th compound will 25 be delivered. For in tance, delivery to a specific si~e of infection may be most easily accomplished by topical application (if the infection is external). Delivery to the cir~ulatory system ~and hence reticuloendothelial cells), may be most easily accomplished by intravenous, 30 intr~peritoneal, intramuscular, or ~ubcutaneous injections.
Since SPLVs allow for ~ sustained release of the compound, doses which may otherwise be toxic to the organism may be utilized in one or more admini~trations to : 35 the organism.
3 Ei77 The ~ections which fol.low de~cr~be ome overall ~chemes in which SPLVS may be u~ed and demons~rate but do no~ limi~ the scope of the pre~;ent invention.
5.302. TREATMENT OF PATHOLOGIES
A number of pathological conditions which OCGUr in man, animal~ and plant~ may be ~reated more effectively by encapsulatiny the appropriate compound or compounds in 10 SPLVs. These pa~hologic condi~ion~ in~lude bu~ are not limited to infections (intracellular ~nd extracellular);
cysts, tumors and tumor cells, allergies~ etc.
~any strategies are possible for using SPLVS in 15 the treatmen~ of such pa~hologies; a few overall ~chemes are outlined below which are particulazly usef~l in that they take advantage of the faot that SPLVs when administered in vivo are internalized by macrophages.
~n one scheme, SPLVs are used to deliver therapeutic agents to sites of intracellular infections.
Certain diseases inYolve an infection of cells of the reticuloendothelical system, e~, brucellosisO These intracellular infections are difficult to cure for a 25 number of reasons: ~l) because the infectious organisms reside within the cells of the reticuloendothelial system, they are sequestered from circulating therapeutic agents which c~nnot cross the cell membrane in therapeutically ~uf~ic~ent concentrations, and, there~ore, are highly 30 resistant to treatment; (2) often the administration of toxic levels of therapeutic agents are required in order to combat such infections; and (3) the treatment has to be ~ompletely effeotive because any residual infection after treatment can reinfect the host organism or can be 35 tran~mitted to sther hosts.
~.i3-According to one m~de of the pre~ent invention, SPLVs containing an appropriate biologically active compound are adminis~ere~ (pre:Eer~bly intr~peritoneally or intravenously) to the ho~t organism or potQntial host 5 or~anism (e.~., in animal herds, ~he uninf~cted animals as wPll as infected animals may be treated)O 5ince phagvcytic cells internalize SPLVs, the admini~tration of an SPLV-encapsulated substance that is biologically active a~ainst the infecting organism will result in directing the bioactive substance to the site o~ infection. Thus, the method o~ the present invention may be used to eliminate infection caused by a variety of microorganisms~
bacteria, parasites, fungi, mycoplasmas, and viruses, including but not limited to: Brucella spp., 15 Mycobacterium ~p~, Salmonella sEp., Listeria pp., F~anci~ella ~ , Hi~topla~ma sp~, Corynebacteri~m spp., Coccidiodes ~e~ and lymphocytic choriomeningitis virus.
The therapeutic agent selected will depend upon 20 the organism causing the infection. ~or instance, bacterial infections m~y be eliminated by encapsulating an antibiotic. The antibiotic can be contained within the aqueous fluid of the 5PLV andJor inserted into the vPsicle bilayer. Suitable antibiotics include but are not limited to: penicillin, ~mpicillin, hetacillin, carbencillin, tetracycline, tetracycline hydrochloride, oxytetracycline hydrochloride, chlortetracycline hydrochloride, 7-chloro-6-dimethyltetracycline, doxycycline monohydrate, methacycline hydrochloride, minocycline hydrochloride, 30 rolitetracycline, dihydrostreptomycin, streptomycin, gentamicin, kanamycin, neomycin, erythromycin, carbomycin, oleandomycin, troleandomycin, Polymyxin B collistin, cephalothin sodium, cephaloridine, cephaloglycin dihydrate, and cephalexin monohydrate.
-.3~-~ e have demon~tra~ed the effec~iveness of ~uch treatment~ in curing brucellosis (~ee Examples, infra).
By ~he procedure of ~his invention~ the effectiveness and duration of action are pro~ong,ed. It i8 surprising that 5 ~his sy~tem i~ effec~ive for treating infe tion~ which do not respond to known treatments suc~ a~ antibiotics entrapped in MLVs~ 5uccessful trea~ment is unexp~cted since any small remaining in~ections will spread and the infectious cycle will commence again. WP have also 10 demons~rated success in ~reating lymphocy~ic choriomeningitis virus infection.
Of oourse, the invention is not limited to trea~men~c of in~cracellular infections. The SPLVs can be 15 ~irected to a~varie~y of sites of infection whether intracellular or extracellular. For inhtance, in ano~her embodiment o~ the prese~t invention, macrophages ~re used to carry an active ~gent to the site of a systemic extracellular infection~ According to this scheme, SPLVs 20 are used to deliver ~ therapeutic subs~ance to uninfected macrophages by administering the SPLVs in vivo (preferably intraperitoneally or intravenously). The macrophages will coalesce with the SPLVs and then become "loaded't with the therapeutic substance; in general, the macrophages will 25 retain the substance for approximately 3 to S days. Once the ~loaded" macrophagec reach the site of infection, the pathogen will be internalized by the macrophages. As a resultJ the pathogen will contact the therapeutic substance contained within the macrophage, and be 30 destroyed~ This embodiment of $he invention is particularly useful in the treatment of Staphylococcus aureus mas~itis in man and cattle.
If the site of infection or a~fliction i5 35 external or ~csessible the SPLV-entrapp~d SherapeuSic _31j_ ~gent can be ~pplied top~cally. ~ particularly u e~ul application involves the treatment of eye afflictions. In the case of ocular a~lictio~ls, SPLVs containing one or more appropria~e active ~ngredien~s may be applied 5 topically to the afflicted eye. A number of organisms cause eye infections in animals and man. Such organisms include but are not limited to: ~oraxell~
Clostridium pp., Corynebacterium ~ , Diplococcus ~p., ~lavobacterium spp~, ~emophilus ~ , Rlebsiella spp., 10 Leptospira ~ r ~ycobacterium ~pp., Nei~seria Propionibacterium ~, Proteus ~ , Pesudomonas ~
Serratia ~ , Escherichia ~ , Staphyloco cus sp~., St~ept~coccus ~eP~ and bacteria-like organisms including Mycoplasma ~ nd Rickettsia ~ These in~ections are 15 difficult ~o eliminate using conven~ional methods because any residual infection remaining after treatmen~ can reinfect through lacrimal ~ecretions. We have demonstrated the use of SPLVs in curing ocular infections caused by Moraxella bovis (see examples, infra).
Because 5PLVs are resistant to clearance and are capable of sustained release of their conten~s, SPLVs are als3 useful in the ~reatment of any affliction requiring prolonged contact with the active treating substance. ~or 25 example, glaucoma is a disorder characterized by a gradual rise in intraocular pressure causing progressive loss of peripheral vision, and, when uncontrolled, loss of central vision and ultimate blindness. Drugs used in the treatment of glaucoma may be applied topically as 30 eyedrops, However, in some cases treatment requires administering drops every 15 minutes due to the rapid cleariny of the drug from the eye socket~ If an affliction such as glaucoma is to be treated by this invention ~herapeutic substances as pilocarpine, 35 ~loropryl, physostigmine, carcholin, aceta~olamide, 6~7 .
ethozol~ide, dichlorphenamide~ carbachol, demecarium bromide~ diisopropylphosphofluoridate t ecothioplate iodide, physostigmine, or neo~tigmine, etc7 can be entrapped within SPLVs which are then applied to the 5 ~ffected eyeO
Other agent~ which may be encapsulated in SPLVs and applied topically include but are not limited to:
mydriatics ~e.~., epinephrine, phenylepinephrineg hydroxy 10 amphetamine, ephedrine, atropine, homatropine, ~copolamine, cyclopentolate, ~ropicamide, encatropine, etc.); local anesthetics; antiviral agent~
~doxuridine, adenine arabinoside, etc.); antimycotic agents (e~y~, amphoteracin B, na amycin, pimaricin, 15 ~lucyto~ine, nystantin, thimerosal, ~ulfamerazine, thiobendazole, tolnh f~ate, ~risiofulvin, etc.);
antiparasitic agents (~ sulfonamide~, pyrimethamine, clindamycin, etc.); and anti-inflammatory agents (e.g., corticosteriods such as ACTH, hydrocortisone, prednisone, 20 medrysone, beta methasone, dexamethasone, fluoromethalone, triamcinalone, etc.).
The following Examples are given for purposes of illustration and not by way of limitation on the ~cope of 25 the inverltion.
6. EXAMPLE: PREPP.RA~ION OF SPLVS
As explained in Section ~.l. the basic method for 30 preparing SPLVs involves dissolviny a lipid or mixture of lipids into an organic solvent, adding an aqueous phase and the mater ial to be ~ncapsulated, and ~onicating the mixture. In the preferred embodiment the solvent is removed during sonication; however, the organic solvent 35 may be removed dur ing or af ter sonication by any ~37-evapora~ive technique. The S~I.Vs u~ed in ~11 of ~he examples contained herein were prepared as described in the following su~sections (however any method which yields SPLVs may be used)~
6.1~ SPLVS CONTAINING ANTIBIOTICS
A 5 mQ diethyl ether solution of lO0 mg leci thin was prepared. The mixture was pla¢ed in a round-bottom 10 flask O Then a solution (0. 3 mQ) containing lO0 mg of s~reptomycin ~ulfate at pH 7 .. d~ in 5 ~ ~IEP~:S
(4- [2 Elydroxyethyl] piperazino 2-ethane sulfonic acid~/0.0725 P~ NaCl/0.0725 M KCl was pipetted into the glass vessel ~ontaining ~he diethyl ether ~olution of 15 lipid. The mixture was placç~d in a bath ~onicator (Laboratory Supplie~ Col. ~ Inc. ) type 10536 for several minutes, (80 kHz frequency:ou~put 80 watts~ while being dried to a viscous paste by passing thereover a gentle stream of nitrogen.
To the viscous paste remaining was added 10 m~ of 5 mM HEPES. The resulting SPLV preparation, containing streptomycin, was suspended in the buffer solution, shaken for ~everal minutes on a vortex mixer, and freed of 25 ~on-encapsulated streptomycin by centrifuging at 12,000 x g for 10 minutes at 20~C. The resulting cake was suspended in 0.5 mQ of 5 mM HEPES.
The procedure described above was followed except 30 that strep~omycin was substituted by each one of the following. dihydrostreptomycin, gentamycin sulfate, ampicillin, tetracyline hydrochloride, and kanamycinO
6~
-3~-6,20 SPLVS CO~TAINING CTHER ~EMBR~NE C~NSTI~UENTS
The proces~ de~cribed in Section 6.1~ was followed ~xcept that any one of ~he following w~s added 5 with ~he egg lecithino (1) phosphatidic acid to give a molar ratio or 8:2 ~lecithin:di~etylphosphate~; (2) stearylamine to give a ~olar r~tio of 8:2 ~lecithin:
~tearylamine); cholest~rol and stearylamine to give a molar ratio of 7:2:1 (lecithin:cholesterol:~tearylamine);
10 and (3) phosphati~ic acid and chole~terol to give a molar ratio of 7:2:1 (lecithin:phosphatidic acid:choles~erol).
6.3. SPLVS CONTAINING PILOCARPINE
The procedure of Section 6cl. wa~ followed except that the antibioti~ ~treptomy~in was replaced with pilocarpine .
~4s SPLVS P~EPARED ~ITH AND WITHOUT BHT
IJndistilled ether contain~ an anti-oxidant, 1 ~g/mQ butylhydroxytoluene (BHT), ~or storage purposes.
The procedure described in Section 6.1. was following using undistilled ether as the solvent in order to 25 incorporate BE~T into the SPLV preparation.
In order to prepare SPLVs without incorporation of BMT, the procedur~ described in Section 6.1. was followed using distilled ether as the solventO
7. EXAMPLE: SPLV MEDIATED DELIVERY IN VITRO
In the following exampl , SPLV mediated delivery of antibiotics to macrophages in culture was demonstrated.
6'7~7 -3~
Peritoneal n~acrc~phage~; were obt~ ed by per itoneal lavage from C5733L~ adult male mice and su~pended in minimal essential medillm (MloE~ pEI 7 1> 2 containing 1096 heat-inactivate~ fetal calf ~erum. Cells 5 were ~u~pended at a concentra~ion oiE 1 ~ 106 cell~ per mQ in 96 well tis~ue s:ul~ure di~hes. To culture~
containing adherent peritoneal macrophages, were added B.
canis at concentraticns of 1 x 106 OEU (colony forming uni~) per m~. After 12 ho~rs, bac~eria no~ engulfed by 10 peritoneal macrophages were removed by repeated washings with ~.E.M. Af er washing of peri~oneal macrophage cul~ures, ~hey were divided into 5 groups, each con~aining 12 replicate cul~ures per group~ ~roup 1, designa~ed Controls~ received no treatmentO Group 2 received aqueous 15 ~treptomycin ~ulfate at a concentration of 1 mg~m~. Group 3 received buffer-filled SPLV~. Group 4 received aqueous ~treptomy~in ulfate (1 mg/mQ) plu5 preformed buffer-filled SPLVs. Group 5 received SPLVs containing treptomycin sulfate (1 mg/mQ)~ After 24 hours, super-natants were removed by repeated washings and peritoneal macrophages were di~rupted by repeated free~ing and thawing. ~erial dilutions of disrup~ed macrophages were plated onto brucella agar and, after 4 days, surviving B.
canis were determined by limiting dilution techniques.
25 Results shown in Table VII indicate that SPLV-entrapped ~treptomycin was totally efective in killing and eliminating the intracellular B. canis infec~ion in vitro.
~he experiment was repeated with Bo abortus 3D exactly as described above except that peritoneal macrophages were obtained by peritoneal lavage from adult female albino guinea pigs. Results are also ~hown in Table ~.1~ I .
TA~LE VII
COLONY-~ORMING ~ S OF INTRA OE LL~LAR
RRUCELLA ISOLAI'ED ~q`~R ~REA~MEN~ OF IN~ECTED
~ACROPHAGES WITH SPLVS CONTAINING STREPTOMYCI~
B~ c~ni~a B. abortusb Controls2.6~1~13x103 3.1+~81x104 Buffer filled~.B2~0.10x1032.9+0.17x104 SPLVs Free 3.11+0.40x103 3.3~0~25x104 StreptomycinC
Streptomycin2.76~0.20x1032.8+0.42x104 15 Plu~ Buffer- .
filled SPLV~C
SP1V-Entrapped 0 0 StreptomycinC
20 a Colony forming units ~CFU) of B. canis ~mean + SD of 12 replicates) i~olated from equal numbers of previously infected mouse (C57Blk) peritoneal macrophages.
b C~U of B. abortus ~mean ~ SD of 12 replicates) isolated from equal numbers of previously infected guinea pi~ peritoneal macrophages.
Concentration of streptomycin 1 mg/mQ.
8. EXAMPLE: TREATMENT INTRACELLULA~ INFECTIONS
The following examples demonstrate how SPLVs can be used in treating intracellular infections. The data presented demons~rate~: (1) the effectivenes~ of using antibiotics encapsulated in SPLV~ in the treatment of disease and ~2) the greater efficiency which is obtained by adminiæteri~g multiple do8es of ~he SPLV prepara~ion.
A compari on of MLVs to SPLYS as vehicles u~ed in the protocols i5 described~
Brucello~is cause~ worldwide economic and public health probl~ms. 2rucellosis is caused by Brucella It is adapted to many mammalian ~pecies, including man5 domestic animals and a variety of wil~ animals~ Six Brucella ~ cause brucellosis in animals; they a~e B.
10 abortus, B. canls, B. melitensis, B neotomae, B~ ovis and _ suis. Both domestic and wild animals serve as reservoirs for potential spread of brucellosis to other animals and man.
Such infections cannot be cleared with antibiotics because the infectious organisms reside within the cells of the reticuloendothelial ystem and are h~ghly resistant to bac ericidal activities of antibio~ics. The quantity of antibiotics reguired and the length of 20 treatment results in either toxic effects on the animal or an unacceptably high concentration of the antibiotic in the tissues of the animal. The further difficulty in treating this disease i~ that the treatment has to be completely *f fective since any remaining infection will ~5 simply spread and the cycle commenceæ once again. The economic impact of such aiseases i~ demonstra~ed by the millions of dollars of valuable cattle which are lost each year due to spontaneous abortion. The only potential way to com~at such infectious outbreaks is to quarantine and 30 then slaughter the animals.
The examples which follow comprise incorporating an antibiotic into SPLVs, and then administrating the encapsulated active substance to the animal~ by 35 inoculating the infected animal~ intraperitoneally.
~2-8Dl~ EFFECT OF A SINGLE ~REATMENT OF B. CANIS
INFEC~ION ~SING ~PLV~ENTRAPPED AN~IBIOTIC
Eighty adult male Swiss mice were infected 5 intraperitoneally (I~P~) with B. cani~ ATCC 23365 ~1 x 107 CFU) and divided i.nto 8 groups of 10 mice ~aci..
Seven days posk-inoculation with B canis~ groups ~er~
~reated as follows: Group 1, ~esignated Controls, received no treatment; ~rQup 2 received bufer-filled 1~ SPLVs (0~,2 m~ I~Po ); Group 3 received aqueou~ streptomycin sulfate (1 mg/kg body weight in a total administration of 0.2 mQ I.P.); Group 4 received aqueous ~treptomycin sulfate (5 mg/kg body weight) in a total admini6tration of 0.2 m~ I. Pr; Group 5 received aqueous streptomycin ~ulfate (10 mg/kg body weight~ in a total admini~tration of 0. 2 mQ
I.P.; Group 6 received SPLVs containing ~treptomycin sulfate (1 mg~kg body weight) in a ~o~al admini~tration of 002 m~ I.P.; Group 7 received SPLVs containing streptomycin sulfate (5 mg/kg body wei~ht) in a total 20 administration of 0.2 m~ I.P.; and Group 8 receive~ SPLVs containing streptomycin ~ulfate ~10 mg/kg body weight) in a total administration of 0.2 m~ I~P~o On day 14 post-inoculation with _ canis, all animals were_ ~acrificed and spleens were removed a~ep~ically. Spleens 25 were homogenized and serially daluted onto bru~,ella agar to determine the number of surviving B. canis in spleer,s after treatment. Results after 4 days incubation are shown in Table VI I I .
3~
t;~
TABLE VIII
fi:F~ECT OF A SINGLE TREA~ME~I~a O~ Bo ~3IS
INFECT~,D !?~5ICE WITH VAR:rOUS CONCENTRATIONS
OF FREE OR SPLV-EMTRA]PPED S~REPTOMYCIN
Colony-l?orming Uni ts B. Canis Per Spleenh No TreatmentBuffer-Filled SPLVs 10~ntrol 3.46x105~2.7x1064.1~slO6~0.66xl36 Streptomycin Concentration Free SPLV-Entrapped (mg/kg bod~ weight)StreptomycinStreptomycin 1 O 5+0 . 4 5x10~1 9 tll+O a 25xlt)3 -- _ 2 ~ 1~+1 . 71~1051 . 52+0 ~ 131~10
The following examples demonstrate how SPLVs can be used in treating intracellular infections. The data presented demons~rate~: (1) the effectivenes~ of using antibiotics encapsulated in SPLV~ in the treatment of disease and ~2) the greater efficiency which is obtained by adminiæteri~g multiple do8es of ~he SPLV prepara~ion.
A compari on of MLVs to SPLYS as vehicles u~ed in the protocols i5 described~
Brucello~is cause~ worldwide economic and public health probl~ms. 2rucellosis is caused by Brucella It is adapted to many mammalian ~pecies, including man5 domestic animals and a variety of wil~ animals~ Six Brucella ~ cause brucellosis in animals; they a~e B.
10 abortus, B. canls, B. melitensis, B neotomae, B~ ovis and _ suis. Both domestic and wild animals serve as reservoirs for potential spread of brucellosis to other animals and man.
Such infections cannot be cleared with antibiotics because the infectious organisms reside within the cells of the reticuloendothelial ystem and are h~ghly resistant to bac ericidal activities of antibio~ics. The quantity of antibiotics reguired and the length of 20 treatment results in either toxic effects on the animal or an unacceptably high concentration of the antibiotic in the tissues of the animal. The further difficulty in treating this disease i~ that the treatment has to be completely *f fective since any remaining infection will ~5 simply spread and the cycle commenceæ once again. The economic impact of such aiseases i~ demonstra~ed by the millions of dollars of valuable cattle which are lost each year due to spontaneous abortion. The only potential way to com~at such infectious outbreaks is to quarantine and 30 then slaughter the animals.
The examples which follow comprise incorporating an antibiotic into SPLVs, and then administrating the encapsulated active substance to the animal~ by 35 inoculating the infected animal~ intraperitoneally.
~2-8Dl~ EFFECT OF A SINGLE ~REATMENT OF B. CANIS
INFEC~ION ~SING ~PLV~ENTRAPPED AN~IBIOTIC
Eighty adult male Swiss mice were infected 5 intraperitoneally (I~P~) with B. cani~ ATCC 23365 ~1 x 107 CFU) and divided i.nto 8 groups of 10 mice ~aci..
Seven days posk-inoculation with B canis~ groups ~er~
~reated as follows: Group 1, ~esignated Controls, received no treatment; ~rQup 2 received bufer-filled 1~ SPLVs (0~,2 m~ I~Po ); Group 3 received aqueou~ streptomycin sulfate (1 mg/kg body weight in a total administration of 0.2 mQ I.P.); Group 4 received aqueous ~treptomycin sulfate (5 mg/kg body weight) in a total admini6tration of 0.2 m~ I. Pr; Group 5 received aqueous streptomycin ~ulfate (10 mg/kg body weight~ in a total admini~tration of 0. 2 mQ
I.P.; Group 6 received SPLVs containing ~treptomycin sulfate (1 mg~kg body weight) in a ~o~al admini~tration of 002 m~ I.P.; Group 7 received SPLVs containing streptomycin sulfate (5 mg/kg body wei~ht) in a total 20 administration of 0.2 m~ I.P.; and Group 8 receive~ SPLVs containing streptomycin ~ulfate ~10 mg/kg body weight) in a total administration of 0.2 m~ I~P~o On day 14 post-inoculation with _ canis, all animals were_ ~acrificed and spleens were removed a~ep~ically. Spleens 25 were homogenized and serially daluted onto bru~,ella agar to determine the number of surviving B. canis in spleer,s after treatment. Results after 4 days incubation are shown in Table VI I I .
3~
t;~
TABLE VIII
fi:F~ECT OF A SINGLE TREA~ME~I~a O~ Bo ~3IS
INFECT~,D !?~5ICE WITH VAR:rOUS CONCENTRATIONS
OF FREE OR SPLV-EMTRA]PPED S~REPTOMYCIN
Colony-l?orming Uni ts B. Canis Per Spleenh No TreatmentBuffer-Filled SPLVs 10~ntrol 3.46x105~2.7x1064.1~slO6~0.66xl36 Streptomycin Concentration Free SPLV-Entrapped (mg/kg bod~ weight)StreptomycinStreptomycin 1 O 5+0 . 4 5x10~1 9 tll+O a 25xlt)3 -- _ 2 ~ 1~+1 . 71~1051 . 52+0 ~ 131~10
9 r 66 3 ~6 ~xlO 41 ~ 32t 1 ~ 00x104 20 ~ I.P. injection in total of 0.2 mQ (sterile saline).
b Surviving B., canis was determined as the number o~
CF11 isolated per spleen and is expressed as mean +
S.D. o~ 10 animals per experiment (triplicate ~xper iments) .
8 0 2 0 EF~ECT OF MULTIPLE TREATMEN~ OF B CAN IS
INFE(:TION l)SIMG SPLV-~NTRAPPED ANTIBIOTIC
Eighty adult male Swiss mice were infected with 30 ~. canis ATCC 23365 (1 x 10 CFI~, I.P. ) and divided into 8 grollps of 10 mice each. Seven days and 10 days post-inoculation with Bo canis, groups were treated as follows~ Group 1, designated contr~ls~ received no trea'cment; ~;roup 2 received buffer-filled ~;PLVs (0.2 mQ, 35 I~IPo ~; Group 3 recei;red aquecus str@ptornycin ~ulfate ~1 ~3~6t~
-~14-~g/kg b~dy weight) in a total ~ldministration of 0.2 mQ, I.P~; Group 4 received a~ueous ~rep~omycin sul~ate (5 mg/kg body weight) in a ~otal admini~tration of 0.2 mQ, I~P.; Group 5 received aquesus ~treptomycin ~ulfate (10 5 mg/kg body weight~ in a total administration o 0.2 mQ, I.P,; Group 6 receiYed SPLVs containing _tr~ptomycin sulfate (1 mg/kg body weight) in a total a~ministration of 0~2 m~, IoP~; Group 7 received SPLVs containing 6treptomycin sul~ate (5 mg/kg body wei~ht) in a total
b Surviving B., canis was determined as the number o~
CF11 isolated per spleen and is expressed as mean +
S.D. o~ 10 animals per experiment (triplicate ~xper iments) .
8 0 2 0 EF~ECT OF MULTIPLE TREATMEN~ OF B CAN IS
INFE(:TION l)SIMG SPLV-~NTRAPPED ANTIBIOTIC
Eighty adult male Swiss mice were infected with 30 ~. canis ATCC 23365 (1 x 10 CFI~, I.P. ) and divided into 8 grollps of 10 mice each. Seven days and 10 days post-inoculation with Bo canis, groups were treated as follows~ Group 1, designated contr~ls~ received no trea'cment; ~;roup 2 received buffer-filled ~;PLVs (0.2 mQ, 35 I~IPo ~; Group 3 recei;red aquecus str@ptornycin ~ulfate ~1 ~3~6t~
-~14-~g/kg b~dy weight) in a total ~ldministration of 0.2 mQ, I.P~; Group 4 received a~ueous ~rep~omycin sul~ate (5 mg/kg body weight) in a ~otal admini~tration of 0.2 mQ, I~P.; Group 5 received aquesus ~treptomycin ~ulfate (10 5 mg/kg body weight~ in a total administration o 0.2 mQ, I.P,; Group 6 receiYed SPLVs containing _tr~ptomycin sulfate (1 mg/kg body weight) in a total a~ministration of 0~2 m~, IoP~; Group 7 received SPLVs containing 6treptomycin sul~ate (5 mg/kg body wei~ht) in a total
10 administration of 0.2 mQ, I.P.; and Group 8 received SPLVs containing streptomycin sulfate (10 ~g/kg body weight) in a total administration of 002 m~, I.P. On day 14 post-inoculation with B. anis, all animals wer~
sacrificed and spleens were removed a~eptically. Spleens 15 were homogenized and ~erially diluted on~o brucella agar to dekermine the number of surviving B. c nis in spleen~
after treatment. Results after 4 days incubation are shown in FIG. 5.
The results of various two-stage treatment regimens on B. canis infections in vivo presented in FIG.
5, demonstrate that in groups receiving aqueous ~treptomycin 7 and 10 days post-inoculation, very little reduction in surviv~ng B. canis in spleens was observed.
25 Only in groups receiving SPLV-entrapped streptsmycin at a concentration of 10 ~g/k~ body weight administered on aay 7 and 10 post-inoculation were all viable bacterial eliminated from spleens of infected animals.
In addition to the experiment described above, various tissues from B. canis infected mice after ~wo treatments with SPLV-entrapped ~treptomycin were sample~
~s follows:
6~
Thirty adult male Swi~s mice w~re inoculated with B. canis ATCC 23365 (1 x 107 CI~U~ I.P.~ ven days post-in~culation animals were divided into 3 group~ of 10 mice each. ~roup 1, designated control~, r~ceived no 5 trea~ment; Group 2 received (on days 7 and 10 post-inocul~tion) aqueous streptomycin ~ulfat~ mg/kg bvdy weight) in each admini~tra~ion of 0.2 mQ~, I.P~;
Group 3 received (on day~ 7 and 10 post-inoculation) SPLVs containing streptomycin sulfate (10 mg/kg body weiyht) in 10 each admini~tration of 0.2 mQ, IoP~ On days 14 to 75 post-inoculation with B. canis, ~11 animals were sacrificed and the following organs removed aseptically, homogenized and serially diluted onto brucella agar for isolation of B. can~s: heart, lungsa ~pleen, liver~
15 kidneys, testes. After 4 days incubation, results of surviving B~ ani~ per organ are shown in FIG. 6.
Results of samplings of various tissues in B.
canis infected mice after two treatment regimens with 20 streptomycin presented in FIG. 6, demonstrated ~hat in animals treated with SPLV-entrapped streptomycin, all tissue~ ~ampled from 14 to 75 days post-inocula~ion with B. canis were totally free of any viable B. canis organisms. In animals untreated or treated with aqueous 25 8treptomycin in concentrations and administration schedules identical to those receiving SPLV-entrapped s~reptomycin, viable B~ canis organisms could be isolated in all tis~ues sampled from 14 to 75 days post-inoculativn with B. canis.
8 . 3 . EFFECTIVENESS OF TREAI~EN~S llSING
~LVS AS COMPARE;D TO SPLVS
Fi~teen adult male Swi~s mice were inoculated 35 with B. canis ATCC 23365 (1 x 107 CFU, I.P.). Seven ~9~16~7~
-~6 days post-~n~cul~tion ~nima~s ~e~re diYi~ed into 3 groups o~ 5 mice each. Group 1, de~ignat d Control~, reoeived no tre2tment; Group 2 reoei~ed (on days 7 ~nd 10 po~t-inoculation) MLVs containing streptomycin ~ul~te (10 5 ~g/kg body weight, I~P.). M~Vs were prepared by conventional techniques using 100 mg egg phosphatidylcholine (EPC~ and 2 m~ of ~terile ~EPES
containing s~rep~omycin sulfate (100 mg/mQ~. The lipid ~o streptomycin ~ulfate ratio wa~ 100 mg EPC to 2~ mg 10 ~treptomycin sulfate in the 2 mQ ~inal M~V ~uspension;
Group 3 recei~ed (~n days 7 ~nd 10 pos~-inoculation) SPL~s containing streptomycin sulfate (10 mg/kg body weight, I~PO ) prepared as desrrib~d in Section 6,10 with the following modifications: 100 mg EPC were used, and 0.3 mQ
15 of ~EPES containing 100 mg 8treptomycin 8ulfate. The lipid to ~treptomycin ~ulfate ratio in SPLVs was lû0 mg EPC to 28 mg ~treptc>mycin ~ulfate in a 2 mQ final s~pensic>n~ On day 14 post-inoculation with B~ can~, all animals were sacrificed and spleens were removed 20 aseptically, homogenized and ~erially diluted onto brucella agar for i~olation of B. canis. Results of ~urviving B~ canls per organ after 4 days incubation are shown in Table IX.
8.4. EFFECT OF VARIO~S SPLV-EN~RAPPED
ANTIBIOTICS ~N TREATMEN~ OF INFECTION
Fifty adult male Swiss mice were inoculated with _ canis ATCC 23365 (1 x 10 CF~, I.P.). Seven days 30 post-inoculation, animals were divided into 10 groups of 5 mice each. Group 1, designated controls, received no treatment; ~roup 2 received buffer-filled SPLVs ~0~2 m~, I~Po ) on days 7 and 10 post-inoculation; GEOUP~ 3, 4, 5 and ~ received aquev~s injections (0.2 m~ I.P.) of 35 diny~ros~reptomycin, gentamycin, kanamycin or ~treptomYcin r~
~7~
T~BLE IX
5COMPARISON OF MLYS AN~D S~LVS CONT~INrNG
STREPTOMYCXN SUL~ATE ON XILLING OF
B. CANIS IN VIVO A~TE'R TWG T~EA~M~NTSa Colony-~orming Unik B. Canis per Spleenb Control 2~7 l.0xlO4 MLVsC l,8+0.4xl04 SPLVs~ o 15 a Intraperitoneal injections, l0 mg/kg body weight, were 6paced a~ 3 day interval~D Controls r~ceived no t~eatment.
b Surviving B~ canis was determined as the number of CF~ isolated per ~pleen and is expressed a~ the mean ~ S.D. of 5 animals per gr~up (duplica~e determirlations per animal~
c Egg pho~phatidylcholine to streptomycin sulfate ratios were l0Q mg lipid to 28 mg streptomycin sulfate.
l0 mg/kg body weight, I.P. on days 7 and l0 post~inoculation tN.B. Each of these antibiotics have been shown to kill B. canis in vitro~. Groups 7, B, 9, and l0 received SPLVs oontaining dihydrostreptomycin, gentamicin, kanamycin, or str2ptomycin at l0 mg/kg body weight on days 7 and l0 postinoculation. ~n day 14 post-inocula~ion with B. canis, all animals were &acrificed and ~pleens w~re removed aseptically7 hom~genized an~ serially diluted onto brucel~a agar for at isolation of B. canis. Result~ of surviving B~ canls per organ after 4 days incubation are as shown in Table X.
~ILE X
COMPARISC!~ OF VARIOUS ANTIBIOTICS ON ~ILLrNG
5:~F B. CAN IS I~ VIYO A~ R TWO TREATMENTSa C~1On~-For~in~ Units B. Canis P~r Spl~en~
Aqueous SPLV-Entrapped So1utions Antibiotic t~ntreated3.93+1.,~1x106 4.66-t0.87x105 Antibiotics~
Dihydrostreptomycin 1O13+0O30x105 0 aTrl~cin7.06+2.53x105 0 Kanamycin 2.72~0q 91x105 0 '~treptomyc in1. 01~0 .17x10 5 0 a Intraperitoneal treatments, 10 mg/kg body weight, :20 were sp~ced at 3 day interva1s. Controls received no ~reatment .
b Surviving B. canis per organ s!~as determined as the number of CFU i~olated per spleen and expressed as the mean + 5.D. of 5 animals per group~ lduplicate determinations per anima1).
25 c Antibiotics effective in ki11ing ~. canis in uspension culture.
The results from tests of various antib~otics on 30 B. anis infected mice presented in Table X demonstrate that an~ibio~ics which are effective in killing B,. canis ln vitro (i~e., in suspension cu1t.~re) are also only effective in ki11ing B. can~s inecltions in ~iYo when they are enc~?s~1ated within SPLVs. Animals receiving either ~gueou~ an~ibi~ties, buffer-filled SPLV~ or no reatmen~
were ln no ~a~e cleared o~ ~urviving B. c~nis in i~ol~ted ~pleen tis~ues.
8.5. TREA~MEN~ OF DC~GS INFECTED WITH }3~ ANIS
~ dult female beagles were in~culat~d with B.
canls ATCC 23365 (l x lO CFU) orally and vaginally~
Seven days po~t inoculation dogs were diYided into 3 groups. Group l~ designated control, received no treatment; Group 2 received (on days 7 and lO
post-in~culation) aqueous streptomycin ul~ate at lO mg/kg body weight (each administra~ion was 5.0 mQ, I~Pr ) ~ Group 3 receiv2d (on days 7 and lO post-inoculation~ ~PLVs 15 con~aini~g ~treptomycin ~ulfate at lO ~g/kg body weight (each administration waC 3O0 mQ, I~P~o Vaginal swab~ings of dogs and heparini2ed blovd ~amples were collected at regular intervals before, during, and at the termination of the ~tudy. These were cultured on brucella agar in 20 order to isolate B. anis. Result6 are ~hown in ~able XI. Serum samples were collected before, during, and at the termination of the study ~or determinations of serum antibody again~t B. canis. These re~ults are al50 ~hown in Table XI. Twenty-one days post-inoculation with B.
25 canis, all animals were euthanized. The following tissues were removed aseptically, homogenized and ~erially diluted onto brucella agar for i~olation of B~ canis: heparinized blood, vaginal exudate, lungs, ~pleen, ~ynovial fluid, uterus, ovary, popli~eal lymph nodes, ~alivary glands, 3~ tonsils, medias~inal lymph nodes, mesenteric l~mph nodes, bone marrow, superficial cervical lymph n~des, and auxiliary lymph nodes. Results of surving B~ canis per tissue after 4 days incubation are ~hown in Table XII.
~q~ 7 --so ~
TABLE XI
5RESUL~S O~ CUL,T~RES AND SER~LOGICAL TESTING
IN B. C~NIS IM~ECTED DO~S SUBJECT~D
TO A TWO TRÆATMENT ANTIBIOTIC REGIMEN a ~ays After }nf~ction SPLV-with b Entrapped c B. Canis Control Streptomycin Streptomycin R P~ B V P~ M B V R M B V
Pr e~treatmen~
O O O O O O O O O O O O O
2 ~D ND ~ -~ ND ND + 0 ~D ND t 4 ND ND ~ ~ ND ND ~ + ND ND ~ +
Post-treatment B 0 0 0 ~ 0 0 ~ 0 0 0 0 0 ~.0 ~ O O + O O O ~ O O O O
~1 1,5 2 ~ ~ 1 2 ~ + 0 0 0 0 a R (rapid slide agglutination test) indicates the reci~rocal of serum titer to B. cani~ antigen (xlO~; 0 = no detectable ~iter~
M (2-mercaptoethanol tube agglutination test) indicates the reciprocal of serum titer to B. canis antigen (x102), 0 - no detectable titer~
In B (blo~d culture) and V (vaginal culture) on brucella agar: ~ ~ detection o greater than or equal to 1 CFU; 0 c no colonies detected. Controls received nc treatment, Streptomycin sulfate (aqueous) 10 mg/kg body weight, I.P.
c SPLVs containing ~treptomycin 6ulfate 10 ~g/kg body weight, I.P.
~ g -5.1 TP,E~LE XII
RISSULTS O~ CULTI~RES i~ROM ~I55UE 5AMPLES
5~[N B . C~l IS IN~ECTEI~ DOGS SUBJECTED
TO A ~O ~RE ATI~;EN T 2~ T I B ~ 0~ I C RE G LQ~N
~PLY~
b Containing c d e Tissue Streptomycin S~reptomycirl Contrvl Whole blQod 9 Vag:Lnal swab 0 Lun~3 s o + ~
Spleen o +
5yn~3vial f lui~ N. 1). 0 0 Ute r u~ 0 Ovary 0 + +
Pop:L i teal lymph node N . D ,, + +
20 Sal;ivary gland o ~ 0 ~onlsil O +
Med.ias tinal lymph node 0 2i . D .
Mesenter ic lymph node . N . D. 0 25 B~,n,e m~lrrow Superficial N.D. N.l).
cer vical lymph node Axillary lymph ns:~de 0 a Animals treated on day 7 and l0 post-infection.
Samples taken ~t necropsy were serially diluted on brucella agar; ~ ~ equal ts:~ or gr~ater than 1 CFU; 0 ~ no colonies.
c 5PLVs containing streptomycin ~ul~ate, l0 mg/kg body weiyht, I.P.
~ Strep'comycir3 ~ulfate 5aqueous) 9 10 mg/kg body wei9ht, I .P.
e Gontrols received no ltreatment.
-52~
~esult~ of culture ~nd ~erologic ~es~s of dogs infected with B. c~nis before, ~urin~, and after two-stage antibiotic adminis~ration are pre~en~ed in Table XI. All animal~ were serologically negative for pre~iou~ exposure to B. canis as measured by neg~ti~e serum t~ter~, ~n~ were ~ulture negative from blood cultures ~nd cultures of vaginal swabbingsO ~11 animals were noted to be culture po~itive for bo~h blood and vaginal cultures prior to treatments on days 7 and 10. Dogs treated with aqueous 10 streptomycin or dogs receiving no treatment remained 2 culture positive for blood and vaginal cultures during post-treatment periods prior to termination on day 21.
Group 3, which received liposomes containing ~reptomycin, became cul~ure negative one day ~f~er the first treatment 15 and remained ne~ative throughout post~trea~ment period~
Dogs which r~ceived no treatment or aqueou~ ~treptsmycin developed detectable serum titers against B. canis antigens by day 21 post-inoculation, while those treated with SPLVs containing antibiotics on days 7 and 10 20 post-incculation did not develop any detec~able an~ibody to ~. canls antigenO
Results from isolation of B. canis from infected d~gs treated with two-stage antibiotic administration 25 which are pres~nted in ~able XII demons rate that in dogs, only treatment with SPLVs containing ~treptomycin was effective in eliminating any viable B. canis in all tissues from all organ samples.
8.6. TREATMEN~ OF B, ABORTUS IN GUINEA PIGS
Fifteen adult female guinea pigs were inoculated with B. abortus A~CC 23451 (1 x 10 CFU, ~.P.). 5even days post-inoculation animals were divided into 3 groups 35 of 5 animals each~ Gro~p 1, designated Controls, received 7'7 o53-no treatmen~. ~roup 2 r~ eived ~gueou~ ~treptomycin~ulfa~e, I.P., injection~ (0.2 ~) at l0 mg/kg bs~dy weight on day 7 and l0 po~ oclalation wi~ch B, ~bor~s. Group 3 received ;PLV~ containin~ ~treptomycin ~ulf~te I . P.
5 injections ~0.2 mQ) ~t l0 ~g/kg bs~dv weight on ~ay~ 7 and 10 post-inoculation with B ~bortl~. On day l4 pc~st-inoculation with :3. abortus, ~ll animals were ~acr ~f iced and ~pleens were removed, a~eptis:ally homogenized and serially diluted onto brucella agar for 10 îsolation of ~. abortus. Results of ~urviving Bo abortus per spleen ~f ter 4 days incubation, ~re shown in ~IG. 7 Only SPLVs containing ~treptomycin were effective in eliminating B. abortus residing within guinea pig ~pleen.
In ~nimals receiving aqueous streptomycin or no treatment, 15 viable B. 2Ibortus bacteria were be identified.
B . 7 . T~ATMENT OF B . ABORTUS INFECTIO~ IN COWS
Nine heavily infec~ed animals were utilized in 20 this experiment. B. abortus bacterial isolations from -milk and vaginal swabbings became and remained negative for six weeks following treatment with SPLVs containing streptomycin. When infection reoccurred in these animals, bacterial isolations were found only in quadrants of the 25 udder which were positive prior to trea~ment.
Nine cross-bred (hereford- jersey-Brangus), 22-month old, non-gravid, confirmed B. abortus culture-positive cowc were used. At least 4 mon~chs prior 30 to ~he ini~iation of the study, ~he animals were experimentally challenged ~ conjunctivum with 1 x 107 CFU o~ bor~us Strain ~3G8 during mid~gestation, which resulted in abortion and/or B, abortus culture po~itive lacteal or uterine secretions and/or fetal tis~ues.
7~
-5~~
C~ws were maintained in ~ndividual i601ation ~talls and ~epara~ed into three groups. ~reatment comprised a two-dose regimen, ~paced 3 ~ays ~p~rt, as follows~ 3 cows were injected in~raper$toneally wi~h 5 physiological ~aline~ (2~ 3 60W5 ~ere injec~d intraperitoneally with aqueous antibiotic (~Sreptomycin at 10 mg/kg body weight) plus preformed buf~er~illed SPLVs~
(3) 3 cows were in~ected intraperitoneally with SPLV-entrapped streptomycin (10 mg/kg body weigh~). The 10 total volume per injection was 100 mQ per animal.
During the firs~ 2 ~onths duplicate bacteriologic cultures of lacteal and uterine secretions were performed weekly providing secretions were obtainable. Then, all 15 cows were euthanatized with an overdo~e of sodium pent~barbitol, and the following organs were ~ollected in duplicate for bacteriologic cultures~ lymph nodes:
left and right atlantal, left and righ~ suprapharyngeal, left and right mandibular, left and right parotid, left 20 and right prescapular, le~t and righ~ prefemoral, left and righ~ axillary, left and righ~ popliteal, left and right intexnal iliac, left and right supramammary, le~t and right renal, bronchial/ ~ediastinal~ mesenteric, ~nd hepatic, (2) glands: all four quarters of mammary gland, 25 left and ri~ht adrenal glands and thymus (if present);
(3) organs and other ~issuesO spleen, liver, left and right horn of ~terus, cervix, vagina, kidney and tonsil.
After necropsy, all tissues were frozen and 30 maintained at -70C whi~e in transport. Tissues were thawed, alcohol flamed, and aseptically tlimmed prior to weighing. Once weight~ wère recorded (0.2 ~o 1.0 grams), the tissue was homogenized in 1 m~ of 6terile ~aline and serially diluted with steril~ saline to 1:10 10 of 35 initial homogenate 6uspension. Aliquot5 (20 ~Q) of each ~5~-dilutio~ from ~eria~ ~uspensions were pla~ed onto brucella agar and pla~e~ in 37C incuba~:lon. Duplicate de~ermi~a~ions were performed 3or ¢ach ti~8ue.
Plates were read dally and ~cored for bacterial yr owth . All colon ies ~ppear ing pr ior to 3 tiay~ w~re i~ola'ced, pa~aged, and gram 6tained to d~termine iden'city. On day~ 5, 6 ~nd 7 during incuba~ion colo~ies with morphology, grswth, and gram staining characteristics 10 consistent with B abortus were counted; the CF~ per gram tissue was then determined. Representative colonies ~ere repas~a~ed for bacterial confirmation of B. abortus.
Bacteriologic isolations were done on all tissue 5 samples and quantitation of bacteria per gram of tizsue were calculated. The re~ults ~rom four ~nimal --one placebo control and three animals treated with SPLV-entrapped ~treptomycin--are presented in Table XIII.
~ 9. EXAMPLE: TREATMENT O~ OCULAR A~LICTIONS
Bacteri~l and like infections as well as many other afflictions of the eye sause worldwide economic and public health problems, leading, if untreated or improperly ~reated, to los5 of .~ight and possible ~eath due to ~epticemia. Bac~erial infeotions of the eye in animals and man have been reported to be caused by a variety of bacteria including but not limited to:
Clostridiu~ spp., Corynebacterium spp., Leptospira spp., 30 Moraxella ~ Mycobacterium spp., Neisseria p~, Propionibacterium ~e~, Proteus s~, Pseudomonas ~2~t Serratia ~ ~, E. Coli ~ , Staphylo~occus ~
Streptococcus ~p~ and ~acteria~like organisms including Mycoplasma ~ and Ricket~sia ~p. Both ~nimals and man 35 ~erve as reservoirs for potential ~pread o~ ~n~ctious bacteria to each other.
~5 -TABLE X I I I
RES~JLTS OF Ct)LTl~RE,S I~RO~5 TISSUE
SAM PLE S OF E~ . ABORI~US IN P`ECTED Co~a S
l~ntreated SPLV-Entrapped ~;tr@ptomycin Ti sgue Corl tr ol 1 ~ 3 Adrenal gland I. 0 0 0 O
Ad~erlal gland R ++ o O
Atlantal I~ R ++ ~ o Atlantal LN L 0 0 5 +
10 P.xillary LN R ~+ 0 ~ O
Axillary LN L ~+ 0 ~ O
Br onchial T!N 0 0 0 0 Cervix 0 0 0 0 E~epa tic LN ~++~ 0 0 ~orn of Uterus L 0 0 0 +
Horn of U'ceru~ P< 0 0 0 O
Int. Illiac LN R ~+ 0 0 O
~n~. Illiac LN L t~ 0 ~ O
Kidney 0 0 0 0 Liver 0 0 0 0 Lung 0 0 0 0 Mammary Gland LF 0 + + O
Mammary ~land LR 0 0 0 Mammary Gland RF ~ o ~ o 20 Mammary Gland RR +~ 0 0 0 Mandibular LN R +~+ 0 û O
~andibular LN L ~++ 0 0 0 Mediastinal LN ~+ 0 + 0 Mesenter ic LN ++~ 0 0 0 Parotid LN L ~+ 0 0 O
Parotid LN R ~ 0 0 Popliteal LN L ~ 0 0 0 25 Popliteal LN R + 0 0 Prefemoral LN IJ ~ 0 0 O
Prefemoral I,N R û 0 0 O
Prescapular LN L 0 0 0 Prescapular LN R +~ 0 0 0 Renal LN 0 0 0 Spleen +++ 0 0 0 Supr amasNnary LN L t~ ~ 0 0 SupramamlDary LN R 0 0 a o Suprapharangeal LN L ~ 0 0 0 Suprapharangeal LN R 0 0 0 0 Thymus 0 0 0 0 Vagina ~ 0 0 0 ~5 0 No de~e~table b~cteria by cl~lture ~f 0. 3 - 1 gm of tissue .
Less than 200 colonie~/gm tissue.
More than 3û0 c~lonies/gm~
More than 1, 000 colorlie~/gm ~
~+~ More than l0û,Q00 colonie /gmO
~a~6~r~
Such bacterial infection cannot be trea~ed with antibiotics without lengthy and cumb~r~me tr~atment ~chedules re~ulting in either ~requent treatments, as rapid as every twenty minu~e~ in humans w~h ~me 5 infections~ ~r unacceptably high concentration~ ~ the antibiotic in the tissues7 Curren~ ~reatment ~ethods are difficult for many other reasons. The infectiou~ organism in the ~urface tiss~es of the eye in ~ome cases ~re highly resistant to bactericidal activities of antibiotics, and 10 topical a~ministration o~ antibiotic~ can result in rapid clearing of the drug from the eye ~ocket yiel~ing varying contact times. As a general rule, treatment of eye infection6 has to be completely effective ~ince any remainin~ infection will simply reinfect through lacrimal 15 gecretions and the cycle commences once again. ~urther, in many cases drug concentrations needed to eliminate the infection can cause impairment of ~ision and in certain cases can result in total blindness. The economic impact of such diseases in domestic animals is demonstrated by 20 the millions of dollars which are lost each year since the only potential way to combat ~uch infectious diseases is sustained therapy and quarantine.
The following experiments evaluate the 25 effectiYeness of treatments using free ~ntibiotic in glycerine as csmpared to antibiotic entrapped in SPLVs for M. bovis infections of the eye~
M. bovis causes infectious keratocc>njunctivitis __ 30 ~pink-eye) in cattle~. This condition is characterized by blepharospasm, lacrimation, conjunc~civitis and varying degrees of corneal opacity and ulceration. Adult cows may develop a mild fever with sligh~cly decreased appetite and a decreased milk pr~ductionc Although a nu~ber of 35 antibiotics are effective again~t M~ bovls, they must be ~dmini~tered early and repeated o~t~n by topical application or ~uboonju~tival ~nject~onO
According to the example~ de cribed herein, the 5 e~fectiveness and duration of action o~ the therapeutic ~ubstance are prolon~ed. I~ i6 ~urpri~ing that this ~ytem ix effective with only one or two adminis~rations ~lnce such infections do not re~pond to simple ordinary treatment with antibiotics. The usual treatments often 10 leave small remaining in~ections which reinfect the eye so that ~he infectious cycle will commence again~ unless the infection is completely eradicated by ~umerous repetitions of the treatment.
9.1. TREAIMEN~ OF IN~ECTIOUS KERATOCONJUNCTIVITIS IN MICE
C57 black mice (160 mice~ were divided into groups. One half ~f each ~r~up was exposed ~o ~.V.
irradiation in each eye (in order to create corneal 20 lesions). All animals were then inoculated with M. bovis instilled onto the right eye at concentrations of 1 x 106 bacteria per eye. Twenty-four hours post-inoculati~n all animals ~ere scored for degree of corneal opacity. The eight groups were treatea by topical 25 application of the following to each eye: Groups 1 and 2 received 10 ~Q of SPLV-entrapped ~treptomycin (30 mg/m~);
Groups 3 and 4 received 10 ~Q streptomycin (100 mg/mQ);
Gro~ps 5 and ~ received 10 ~Q of buffer-fil1ed SPLVs ~uspended in aqueous streptomycin (100 mg/mQ); and Groups 3~ 7 and 8 received 10 ~Q of sterile ~aline (N.B. The uninfected left eyes were treated with the same topical solutions in order to determine whether SPLVs would irritate the eye; no irritation was observed). Once daily, animals were scored for progre~sion or regression 35 of corneal lesions ~nd vn days 3, 5 and 7 post-treatment -5g-right eyes were ~wabbed and i~41.~ion8 for MD bovi~ were performed on repre~entative animals~ M. bo~ olonies were de~ermined by colony ~orphology and reac~i~lty to flour@6cently l~beled antibvdy 1:o M. ~ovi~ pil~ J, Re~ults, ~h~wn in Table XIV, re~eal that o~ly the SPLV~entrapped treptomycin was effective in elimina~iny infec'cion..
902~ TREATMENT 0~ RABBIT CONJUNCTIV~
~SING SPLV~ENTRAPPED ANTIBI8TIC
pl~. bovis, ~TCC ~rain 10900, were diluted to a concentration of 1 x 107 cell~ per mQ in ~terile aline t0.08596 NaCl). Aliquots (0.1 mQ) o~ bacterial ~uspensions were inoculated ~opically into the eyes of ten ~dult 15 ~emale rabbit~. Samples ~or cultures were taken daily by 6wabbing the conjunctivae and pla~ed onto blood agar plates ~or isolation of M bo~ls. Three days post-inoculation, rabbits were divided into 3 groups: 2 animals (controls) received no treatment; 4 animals 20 received treptomycin in ~terile saline (concentration lO
mg/kg body weight); and 4 animals received SPL~-entrapped streptomyein in a ~terile saline solution (concentration lO mg streptomycin/kg body weight). ~ll solu~ions were administered 'copically into each eye . Af ter 24 hours, the 25 swabbing of conjunctivae of all rabbits wa~ resumed and continued daily ~or seven daysO The results of isolation for M bovis ~n blood ~gar plates are shown in Table XY.
~ , .
9.3. TREAlMENT 0~ RERATOCONJUNCTIVITIS
~ESULTING ~ROM SUBCUTANEOUS INFECTIONS
M. bovis, ATCC ~train lO900, were diluted to a _ _ _ _ . _ c:oncentration of 1 x 107 cell~ per mQ in l;terile saline~ Aliquots (O.l mQ) of bac~erial su~pensions were 35 inoculated into the eyes of adul~ rabbit6 which had been TABLE XIV
~E SULTS OF TREATMEN ~ OF Ii~d E'E CT IOU
KERATOCONJ[~NCTIVITIS R~S17LTING FiRC~
OCULARs INFECTIC)N~ OF M . BOVIS IN MICi3 M~ Bovis Number of Mice Per Group of 20 Culturesa Pre-Treatmen~ Post-Tr~a'cment Days Post-Corn~al Qpacityb Corneal Opacityb Treatment D l 2 3 4 0 l 2 3 4 3 5 Non-radiated Mice Controls 16 3 0 l 0 18 2 0 0 04/5 4/5 I?r ee S~reptomycinC 18 1 1 0 O lB 2 0 O 02/5 ~/5 BLlf r e r -f i 1led SPLVs pl u s f r ee Streptomycin~ 17 2 l 0 0 18 l l 0 02/j 3/5 SPLV~-En tr apped StreptomycinC 17 3 0 0 0 20 0 0 0 00/5 0/5 W-Radi~ted Mice Controls l l 5 9 4 l0 3 l 2 45/5 5/5 Fr ee StreptomycillC O 4 9 7 0 14 ~ 2 1 03/5 4f5 Buffer~filled SPLVs plus free StreptomycinC 0 3 5l0 2 ll 2 4 3 03/5 3~5 SPLVs - En tr apped StreptomyGinC 0 l 5ll 3 19 l 0 0 00/5 0/5 a Culture c>f eyes positive f~r presence of ~3. bovis, determined by ~luorescent antibody staining.
b Scoring of norlDal cornea: 1 8 loss of normll luster;
~ - small foci o~ opacity; 3 ~ partial opacity Of cornea; 4 = total opacity of cornea.
Total administration l0 llQ ~l,0 mg streptomycin per ~Y~ ) .
~ABLE XV
5RE5ULTS OF ISOL~ION ~OM RAB~IT
CO~JtJNCl~lVAE ~TER ~OPICALLY IN~ECTING
WTTH M BOVIS AND TREATING ~ITH
AQUEOUS OR~PLV-ENCAPSULATED STREPTOMYCIN
M. bo~ Culture~a An imal D~y s P~ t - I n ~ec t i on Group Number Pre-Treatmen-b Post-~reatmentC
3 ~ 5 6 7 Con tr ol l 0 ~ + ~ ~ + +
2 0 ~ ~ + ~ ~ +
Str eptomycind l 0 ~ ~ +
2 0 0 ~ ~ + +
3 ~ ~ ~ +
4 0 ~ + + + ~ +
SPLY Entrapped 1 0 0 ~ O 0 0 O
Str eptomycine 2 0 + ~ 0 0 0 0 3 0 + ~ 0 0 0 4 û + + O 0 0 O
a Cultures scored for presence of M. bovi~ colonies on blood agar plates after 24 hours at 37C. Plus (~) represents greater than or equal to 1 CFU ~3. bovis per i~olate; 0 represent~ no detectable colonies.
25 b All animals inoculated with l x l0S CFU M. bovis topically in each eye.
c Animals ~reated with 0 O l m~ solution topically in each eye~
d Streptomycin (l0 mg/kg body weight) in sterile saline ~olution.
SPLV-entrapped ~treptomycin (l0 mg/kg body weight) in ~terile saline ~olu'Lion.
--6;~o pr~viou~ly infected a~ de~cribec9 in Section 9. 2. ~nd were not treated with SPLVs. The ri~ht eye6 of all nine rabbits were irloculated wi~h 0.:L mQ of M. bovis ~ubcutaneou~ly into conjunctiva:L ti~ue and in the left 5 eyes of ~ll rabbits s~/ere irloculated wi th 0.l m~ of M.
bovis ~opically. Cultures we~e taken daily from __ 6:onjuncti~ae of both eyes from all rabbits And plated onto blood agar plates for isolation of Mo bovis. Three days post-inoculation, rabbits were divi~ed in~o 3 group~: 2 0 animals received no treatment; 3 animals received streptomycin in a ~tandard ophthalmic glycerin ~uspension (concentration of streptomycin lOmg~kg body weight~; ~nd 4 ani~al~ received a 6aline 6u~pension of SPLV-entrapped streptomycin tlO mg of streptomycin ~ulfate per kg of body 15 weight). The.~uspension or 501UtiOTI was a~mini~tered topically (0.l mQ) into each eye. After 24 hour~ ~nd on each o~ the next five days, conjunctiYal swabbings were taken from all rabbits. The results of isolation for M.
bovis on blo~d agar plates are shown in Table XVI.
20 Necropsies were performed on all animals at the termination of experiments and conjunctivae were removed from all animals. These were ~cored for vascularization, and were minced, hom~genized and plated onto blood agar plates for isola~ion of M. bovis. Results are shown in 25 Table XVII.
9 . 4 . EVAL~ATION OF THE EFFECTIVENESS OF SPLVS
AS COMPARED I~O LIPOSOME PREPARATIONS IN
T~: TREATMEMT OF OCULAR INFECTIONS
M. bovis (ATCC strain 10900) were diluted to a concentration of l x l0 ' cells per mQ in 6terile ~aline. Aliquo'cs IOol mQ) of bacterial su~;pensions were inc>culated subcutaneously into the conjunctival tissues of 35 both eyes in adult rabbi~s. Swabbings were ~aken daily 7~
~63-~ABLE XVI
RESI~LTS 0~ ISOLA~ION P`P~OM RP~BBIT CONJUI~Cq`IVAE
AFTER INVCVLA~ION OF Pq ~ BOVIS INTO
~NJUNC~IYAL ~BRANES ~3D TREATMENI g~TH
ST~PTOMYCIN IN OPH~ALMïC GLYOERINE S9I-UTIC
OR SPLV-ENCAPSIJLATE:D STREPTOP~YCIN IN SALINE
P~. bo~i~ Culturesa Day~ Po~ t Infec t i on An imal Group Numberb Pre-treatment Post-Treatment Corl tr ol 1 +
~trep~omyc in l ~ +
in Glycer ine 2 +
solutlon d 3 + ~ ~ +
SPLV~ û O O
Encapsulated 2 ~ + O 0 O
20 Str eptomyc ine 3 + + 0 0 0 ~ 4 ~ ~ ~ 0 0 a Cultures scored for presence of M. bovis colonies on blood agar plates af'cer 24 hour~ ~t 37~C. Plus ~+) represents greater than or equal to 1 CFU M. bovis per i~olate; 0 represents no detectable co~nies.
b All animals were inoculated with 1 x l06 CFU M.
bovis topically in both eyes; l x lO6 CFU M. bovis was lnjected into conjunctival membranes, ln right eyes; and l x lO6 CFU M. bovis was applied topically in le~t eyes.
3o c Animals treated with 0. l mQ solution tc~pically in each eye.
d ~imals treated topically in ~ach eye with 6treptomycin (lO mg/kg body weight~ in t)ph~chalmiC
glycer ine base ., 35 e Ar imals ~reated ~opica:lly in each eye wi~h SPI.V-encapsulaed ~treptomycin ~lO mg~kg bc~dy weight) in sterile ~aline 60lution.
6~7 ~6~-TABLE XVII
RESULTS ~ROM N~CROPSY OF T}~ ORBIT AND AS~OCIATED
TISSUE~ FROM R~BBITS ~FTER ~M~LA~ION WIT~ M~ OVIS
INTO CONJUNCTIYAL TISS~ES ~ND ~EATMENT WIT~ EI~HER
STREPTOMYCIN IN OP~THALMIC ~LY OE RINE SOLU~IOM ~R
SPLV- NCAPS~LATED STREPTOMYCIN I~ STERLINE SALINEa Isol~tion of M. bovis Vascularization Cultures of Right Eye b Control A ~ 2+
B + 2 Streptomycin in 15 ~lycerine Sol~tion A ~ 2+
B + l~
C ~ 2+
D + 2+
20 SPLV-encapsulated S~reptomycin O O
~5 a Legends are same as ~able XII, performed on day 5, post infection.
b Vascularization scored as follows: 0 = vessels normal; l = some vessels definitely dilated and infiltrated by minor vessels; 2 = diffuse red with individual vessels not easily discernible; 3 =
diffuse bee~y red, vascular leakage and effusion of blood into conjunctivae~
-65~
~rom conjunctivae of both eye~ from all rabbits and plated onto blood agar plate~ for i~olation of M~ bovi~. ~ive day~ po~t-inoculation, rabbits were divi~d ln~o 6 groups: 2 animal~ received no tre~tment (contro-l~); 3 - ~ anim~ls ~eceived a ~uspension of SP~V-encapE~la~ed streptomycin (lOmg of s~reptomycin ~ulfa~e per kg of body weight) which when diluted 1~100 had an O~D.4~0 (optical density at 480 nm) equal to 0.928; 3 animals received a suspension of SPLV-encapsulated ~treptomycin (10 mg of ~treptomycin ~ulfa~e per kg of body weight) which when dilu~ed 1:100 had an O~D~480 equal to 0.449; 3 animals received a ~uspension of SPLV encapsulated strep~omycin (10 mg streptomycin ~ulfate per kg of body weight) whi~h when diluted l:lOG had an O.D.480 equal to 0.242; 3 animal~ received a 6~spension of SPLV-encap~ulated ~treptomycin (10 mg 6treptomycin 6ulfate per kg body weight) which when diluted 1:100 had an O.D~80 equal ~o 0.119; and 2 animals received a suspension of multilamellar vesicles (MLVs) containing streptomycin (10 20 mg strep~omycin ~ulfate per kg of body weight) wi~h an O.D.480 of a 1:100 dilution equal to 0~940. MLVS were made ~y the process of ~ountain et al. Curr. Micro. 6:373 ~1981) by adding ~treptomycin ~ulfate to the dried lipid film which was then vortexed, and allowed to swell for two 25 hour s; ~che non-entrapped streptomycin was removed by repeated centr if ~gation.
The suspensions were administered topically into each eye. After 24 hours, conjunctival swabbings were 30 taken from all rabbit~ àaily for g days and plated onto blood agar. The results of isolation for M. bovis on blood agar plates ~re shown in T3ble XVIIIo Necropsies were performed on all animals. These were scored for lacrimal secretions, anà conjunctivae were removed 35 aseptically ~Erom all animals. These were scored for 8~7~9 ~66-~a~ularization~ and were minced, homogen~zed ~nd pl~ted onto blood ~gar plate~ for i~olation of M is~ Result are shown in Table XIX.
10. EXAMPLE: TR~:A~ENT O~ VIRAL IN~ECTIONS
Lymphocytic choriomeningitis viru~ (LCMV)~ ~
member of the Arenaviru~ group, is known to cau~e diseases in man and LCMV infection is fatal in mice inosulated 10 intracerebrally with this virus. The death of mice is caused by the immune cells which react against virus-infected cells. The virus does not kill the cells in which it ~ltiplies, therefore, the therapeu~i~ agent used in mice mu~t either inhibit ~irus multiplica'cion ~o 15 tha~c the inunune cells will not be act~Yati?d, ~nd/~r inhibit the actiYation of immune cell~.
The following example demonstrates the effectiveness of trea~ing viral infections by 20 administering a SPLV-encapsulated antiviral compound.
10.1. ~REATMENT O~ LETHAL LYMPHC~Y~IC CHORIO-~SENINGITIS VIRUS INFECTIONS IN MICE
5wiss mice 2 months of age were inoculated intracerebr~lly with a lethal dose of LCM virus, i ~., lO0 plaq~e forming units (P~U) in 0.05 mQ inoculum per mouse.
Mice were divided into 4 groups of 7 animals each and were ~reated on days 2, 3 and 4 pos~-infection by 30 in~raperitoneal injections with O.l mQ/dose/mouse as follows~ he ~5PLV-R group" was treated with a ~uspension of e99 phs:~sphatidylcholine SPLVs contalning 3 mg Ribavarin/m~. SPLVs were prepared using 100 mg lipids and 0.3 mQ of lO0 mg drug/mQ in PBS buffer; ~he ~ntrapment ~67~
TABLE XVIII
ISOLATION OF M. BOVIS FROM INFEC~ED RABBIT
CONJUNC~IYAE A~TER ~REATMENT ~fT~ DILUTIO~S
0~ SPLV-ENCAPSULATED STREPTOMYCIN I~ ~ALINE
OR MLV-ENCA~S~LATED STREP~OMYCIN IN SALINE
I,olation sf ~. b~v s ~ay~ Po~t~In ect.ol Animal Pre-Treatmen. Post~ eatment Group Number 1 ~ 3 4 6 7 8 9 ....... 11 12 13 14 Co~ol 1 + ~ ~ + ~ + + ~ + +
2 + ~ + + + ~ ~ + + +
MLV-15 encapsulated 1 + ~ + ~ 0 0 + + +
Streptomycin 2 t ~ O ~ ~ O O + ~ O
SPLV-encapsulated 1 + + ~ ~ + 0 0 0 0 0 0 0 0 0 Streptomycin 2 + ~ ~ ~ + 0 0 0 0 0 0 0 0 0 (undiluted) 3 ~ ~ ~ + + 0 0 0 0 0 0 0 0 SPLV-encapsulate~ 1 ~ + + + + ~ 0 + ~ ~ + +
Streptomycln 2 + + ~ ~ ~ 0 D + 0 0 0 0 0 0 (1:2 dilution) 3 ~ ~ + ~ + 0 0 0 0 ~ 0 0 0 0 SPLV-encapsulated 1 + ~ + ~ + + + 0 0 0 0 0 0 0 25 ~treptomycin 2 + + ~ + ~ ~ 0 0 0 + 0 0 0 0 (1:4 dilution) 3 ~ ~ + + + 0 0 + ~ ~ + ~ ~ +
SPLV-encapsulated 1 + ~ ~ ~ + 3 0 ~ + + + ~ ~ +
Strepto~ycin 2 + ~ 0 0 0 0 0 0 0 0 0 ~1~6 dilution) 3 ~ + ~ + + 0 + ~ + ~ +
3~
a All animals inoculated wi~h 1 x 106 C~ M. bovis by injecti~n into conjunctival membranes o~ both eyes.
Conjunctival swabbings were plated on ~l~od agar.
Cultuzes scored for presence of M. ovis colonies on blood agar plates after ~ hours a~ 37~C; + ~ greater than or equal to 1 CFU; 0 = no detect~ble cultures.
TABLE XIX
~ESULT5 FROM NECRO~SY OF ~E~ ORBIT A~D
A~SOCIATED TISSUES FR0M RABBI~S AFTEP~
INOCULATION WIT~ M. BOVIS IN~O CONJUNCTIY~L
~ISSUES AND TREATMEN~-~IT~ EI~HER MLV-ENCAPSULATED
STREP~OMYCIN, SPLV~ENCAPS~LATED STREPTOMYCIN
OR DILVTION~ OF SPLV-ENCAPS~LATED STREPTOMYCINa Isolation Vasculariza~ Lacrimal Animal of M. Bovis tion o~ Eyesb DischargeC
Control l + l+ l+
2 ~ l~ l+
MLV
encapsulated l + l+ l+
15 S~rep~omycin . 2 0 l+ 0 encapsulated l 0 0 0 Strep~omycin 2 0 l~ 0 (~ndiluted) 3 0 0 0 SPLV-20 encapsulated l ~ 2+ 2+
Streptomycin 2 0 0 0 (l:2 dilution) 3 0 l+ 0 SPLV-encapsulated l 0 0 0 Streptomycin 2 0 l~ 0 25 (l:4 dilution) 3 + l+ 0 SPLV-encaps~la~ed l + l~ l+
Streptomycin 2 0 l~ 0 (1:6 dilution) 3 ~ l~ 0 3o a Legends are same as Table XIV, per~ormed on day 14 post-infection.
b Vascularization scored as follows: 0 = ~essels normal, l ~ome vessels dilated and infiltrated by minor vessel~; ~ = diffuse red with individual vessels not easily discernable; 3 - diffuse beefy r~d/ vascular leakage and e~f~sion of blood into conjunctivae.
Discharge ~cored as follows: 0 c no discharge; l =
di~charge with moi~tening of lids ~nd hairs adjacent to lids0 2 = discharge with moistening of lids, hairs ~nd areas ad~acent to eyes.
7~
TABLE XX
6 T~EATMENT O~ LETHAL LCM VIRUS IN~ECTI~W IN ~ICEa Gr oupLe'Lhali~cyCVirus P~ecovered from Splee (PYU x 105~ Q) Control 5/5 7. 0 SPLV-gr oup 5/5 6 ., 9 ~R group 5/5 5O 2 SPLV-R Gr oup 3/5 3 ~ 4 Two month old mice were each inoculated intracerebrally with a lethal dose~ lO0 PFU of LCM viru~ in 0.05 mQ inocula.
b Lethality is expressed as number dead/number in group.
c On the fi~th day post~infection 2 mice from each group were sacrificed and their spleens homogenized at a concentration o~ l gm ~pleen/20 mQ homogenate.
of drug was 10~; (2) the ~R-groupe' was treated with a ~olution of Ribavarin 3 mg/m~ in PE~S; (3) the "SPLV-~roup"
25 was ~rea~ed wi~h buffer~filled 5PLVs (i.e~, SPLVs prepared a~ above bu~ without Ribavarin); and ~4) the "control group" was tre~ted with PBS. On day 5 post-infection mice from each group were sacrificed and their spleens homogen i zed ( 2 6pleen s~gr oup wer e homog2n i zed in PBS a t 3~ 1/20 weight per volume buffer). 'rhe plaque ~orming units (PFU~ per mQ were determined fs?r each suspension~ The remaining 5 mice in each groups were obserYed for lethality two ~:ime~ daily for 30 day~. The re~;ul'cs ~re presented in Table XX.
~5 --7 ~--~ rable XX clearly indica~e~ a decrease in lethali~y and a ~3ecrease in th~ viru~ recoverable ~rom the ~nfected animal~ We have nst ye~c determined whe~her ~he~e resul~s are due ~co the anti-viral wtlvi~y o~ the 5 ribavarin which i~ relea~ed from the SPLYs i~r whether it i~ due to an i~ununomodulation of the mouse ho~t durin~ the su6tained release of rib~varin ~rom the SPLVs.
sacrificed and spleens were removed a~eptically. Spleens 15 were homogenized and ~erially diluted on~o brucella agar to dekermine the number of surviving B. c nis in spleen~
after treatment. Results after 4 days incubation are shown in FIG. 5.
The results of various two-stage treatment regimens on B. canis infections in vivo presented in FIG.
5, demonstrate that in groups receiving aqueous ~treptomycin 7 and 10 days post-inoculation, very little reduction in surviv~ng B. canis in spleens was observed.
25 Only in groups receiving SPLV-entrapped streptsmycin at a concentration of 10 ~g/k~ body weight administered on aay 7 and 10 post-inoculation were all viable bacterial eliminated from spleens of infected animals.
In addition to the experiment described above, various tissues from B. canis infected mice after ~wo treatments with SPLV-entrapped ~treptomycin were sample~
~s follows:
6~
Thirty adult male Swi~s mice w~re inoculated with B. canis ATCC 23365 (1 x 107 CI~U~ I.P.~ ven days post-in~culation animals were divided into 3 group~ of 10 mice each. ~roup 1, designated control~, r~ceived no 5 trea~ment; Group 2 received (on days 7 and 10 post-inocul~tion) aqueous streptomycin ~ulfat~ mg/kg bvdy weight) in each admini~tra~ion of 0.2 mQ~, I.P~;
Group 3 received (on day~ 7 and 10 post-inoculation) SPLVs containing streptomycin sulfate (10 mg/kg body weiyht) in 10 each admini~tration of 0.2 mQ, IoP~ On days 14 to 75 post-inoculation with B. canis, ~11 animals were sacrificed and the following organs removed aseptically, homogenized and serially diluted onto brucella agar for isolation of B. can~s: heart, lungsa ~pleen, liver~
15 kidneys, testes. After 4 days incubation, results of surviving B~ ani~ per organ are shown in FIG. 6.
Results of samplings of various tissues in B.
canis infected mice after two treatment regimens with 20 streptomycin presented in FIG. 6, demonstrated ~hat in animals treated with SPLV-entrapped streptomycin, all tissue~ ~ampled from 14 to 75 days post-inocula~ion with B. canis were totally free of any viable B. canis organisms. In animals untreated or treated with aqueous 25 8treptomycin in concentrations and administration schedules identical to those receiving SPLV-entrapped s~reptomycin, viable B~ canis organisms could be isolated in all tis~ues sampled from 14 to 75 days post-inoculativn with B. canis.
8 . 3 . EFFECTIVENESS OF TREAI~EN~S llSING
~LVS AS COMPARE;D TO SPLVS
Fi~teen adult male Swi~s mice were inoculated 35 with B. canis ATCC 23365 (1 x 107 CFU, I.P.). Seven ~9~16~7~
-~6 days post-~n~cul~tion ~nima~s ~e~re diYi~ed into 3 groups o~ 5 mice each. Group 1, de~ignat d Control~, reoeived no tre2tment; Group 2 reoei~ed (on days 7 ~nd 10 po~t-inoculation) MLVs containing streptomycin ~ul~te (10 5 ~g/kg body weight, I~P.). M~Vs were prepared by conventional techniques using 100 mg egg phosphatidylcholine (EPC~ and 2 m~ of ~terile ~EPES
containing s~rep~omycin sulfate (100 mg/mQ~. The lipid ~o streptomycin ~ulfate ratio wa~ 100 mg EPC to 2~ mg 10 ~treptomycin sulfate in the 2 mQ ~inal M~V ~uspension;
Group 3 recei~ed (~n days 7 ~nd 10 pos~-inoculation) SPL~s containing streptomycin sulfate (10 mg/kg body weight, I~PO ) prepared as desrrib~d in Section 6,10 with the following modifications: 100 mg EPC were used, and 0.3 mQ
15 of ~EPES containing 100 mg 8treptomycin 8ulfate. The lipid to ~treptomycin ~ulfate ratio in SPLVs was lû0 mg EPC to 28 mg ~treptc>mycin ~ulfate in a 2 mQ final s~pensic>n~ On day 14 post-inoculation with B~ can~, all animals were sacrificed and spleens were removed 20 aseptically, homogenized and ~erially diluted onto brucella agar for i~olation of B. canis. Results of ~urviving B~ canls per organ after 4 days incubation are shown in Table IX.
8.4. EFFECT OF VARIO~S SPLV-EN~RAPPED
ANTIBIOTICS ~N TREATMEN~ OF INFECTION
Fifty adult male Swiss mice were inoculated with _ canis ATCC 23365 (1 x 10 CF~, I.P.). Seven days 30 post-inoculation, animals were divided into 10 groups of 5 mice each. Group 1, designated controls, received no treatment; ~roup 2 received buffer-filled SPLVs ~0~2 m~, I~Po ) on days 7 and 10 post-inoculation; GEOUP~ 3, 4, 5 and ~ received aquev~s injections (0.2 m~ I.P.) of 35 diny~ros~reptomycin, gentamycin, kanamycin or ~treptomYcin r~
~7~
T~BLE IX
5COMPARISON OF MLYS AN~D S~LVS CONT~INrNG
STREPTOMYCXN SUL~ATE ON XILLING OF
B. CANIS IN VIVO A~TE'R TWG T~EA~M~NTSa Colony-~orming Unik B. Canis per Spleenb Control 2~7 l.0xlO4 MLVsC l,8+0.4xl04 SPLVs~ o 15 a Intraperitoneal injections, l0 mg/kg body weight, were 6paced a~ 3 day interval~D Controls r~ceived no t~eatment.
b Surviving B~ canis was determined as the number of CF~ isolated per ~pleen and is expressed a~ the mean ~ S.D. of 5 animals per gr~up (duplica~e determirlations per animal~
c Egg pho~phatidylcholine to streptomycin sulfate ratios were l0Q mg lipid to 28 mg streptomycin sulfate.
l0 mg/kg body weight, I.P. on days 7 and l0 post~inoculation tN.B. Each of these antibiotics have been shown to kill B. canis in vitro~. Groups 7, B, 9, and l0 received SPLVs oontaining dihydrostreptomycin, gentamicin, kanamycin, or str2ptomycin at l0 mg/kg body weight on days 7 and l0 postinoculation. ~n day 14 post-inocula~ion with B. canis, all animals were &acrificed and ~pleens w~re removed aseptically7 hom~genized an~ serially diluted onto brucel~a agar for at isolation of B. canis. Result~ of surviving B~ canls per organ after 4 days incubation are as shown in Table X.
~ILE X
COMPARISC!~ OF VARIOUS ANTIBIOTICS ON ~ILLrNG
5:~F B. CAN IS I~ VIYO A~ R TWO TREATMENTSa C~1On~-For~in~ Units B. Canis P~r Spl~en~
Aqueous SPLV-Entrapped So1utions Antibiotic t~ntreated3.93+1.,~1x106 4.66-t0.87x105 Antibiotics~
Dihydrostreptomycin 1O13+0O30x105 0 aTrl~cin7.06+2.53x105 0 Kanamycin 2.72~0q 91x105 0 '~treptomyc in1. 01~0 .17x10 5 0 a Intraperitoneal treatments, 10 mg/kg body weight, :20 were sp~ced at 3 day interva1s. Controls received no ~reatment .
b Surviving B. canis per organ s!~as determined as the number of CFU i~olated per spleen and expressed as the mean + 5.D. of 5 animals per group~ lduplicate determinations per anima1).
25 c Antibiotics effective in ki11ing ~. canis in uspension culture.
The results from tests of various antib~otics on 30 B. anis infected mice presented in Table X demonstrate that an~ibio~ics which are effective in killing B,. canis ln vitro (i~e., in suspension cu1t.~re) are also only effective in ki11ing B. can~s inecltions in ~iYo when they are enc~?s~1ated within SPLVs. Animals receiving either ~gueou~ an~ibi~ties, buffer-filled SPLV~ or no reatmen~
were ln no ~a~e cleared o~ ~urviving B. c~nis in i~ol~ted ~pleen tis~ues.
8.5. TREA~MEN~ OF DC~GS INFECTED WITH }3~ ANIS
~ dult female beagles were in~culat~d with B.
canls ATCC 23365 (l x lO CFU) orally and vaginally~
Seven days po~t inoculation dogs were diYided into 3 groups. Group l~ designated control, received no treatment; Group 2 received (on days 7 and lO
post-in~culation) aqueous streptomycin ul~ate at lO mg/kg body weight (each administra~ion was 5.0 mQ, I~Pr ) ~ Group 3 receiv2d (on days 7 and lO post-inoculation~ ~PLVs 15 con~aini~g ~treptomycin ~ulfate at lO ~g/kg body weight (each administration waC 3O0 mQ, I~P~o Vaginal swab~ings of dogs and heparini2ed blovd ~amples were collected at regular intervals before, during, and at the termination of the ~tudy. These were cultured on brucella agar in 20 order to isolate B. anis. Result6 are ~hown in ~able XI. Serum samples were collected before, during, and at the termination of the study ~or determinations of serum antibody again~t B. canis. These re~ults are al50 ~hown in Table XI. Twenty-one days post-inoculation with B.
25 canis, all animals were euthanized. The following tissues were removed aseptically, homogenized and ~erially diluted onto brucella agar for i~olation of B~ canis: heparinized blood, vaginal exudate, lungs, ~pleen, ~ynovial fluid, uterus, ovary, popli~eal lymph nodes, ~alivary glands, 3~ tonsils, medias~inal lymph nodes, mesenteric l~mph nodes, bone marrow, superficial cervical lymph n~des, and auxiliary lymph nodes. Results of surving B~ canis per tissue after 4 days incubation are ~hown in Table XII.
~q~ 7 --so ~
TABLE XI
5RESUL~S O~ CUL,T~RES AND SER~LOGICAL TESTING
IN B. C~NIS IM~ECTED DO~S SUBJECT~D
TO A TWO TRÆATMENT ANTIBIOTIC REGIMEN a ~ays After }nf~ction SPLV-with b Entrapped c B. Canis Control Streptomycin Streptomycin R P~ B V P~ M B V R M B V
Pr e~treatmen~
O O O O O O O O O O O O O
2 ~D ND ~ -~ ND ND + 0 ~D ND t 4 ND ND ~ ~ ND ND ~ + ND ND ~ +
Post-treatment B 0 0 0 ~ 0 0 ~ 0 0 0 0 0 ~.0 ~ O O + O O O ~ O O O O
~1 1,5 2 ~ ~ 1 2 ~ + 0 0 0 0 a R (rapid slide agglutination test) indicates the reci~rocal of serum titer to B. cani~ antigen (xlO~; 0 = no detectable ~iter~
M (2-mercaptoethanol tube agglutination test) indicates the reciprocal of serum titer to B. canis antigen (x102), 0 - no detectable titer~
In B (blo~d culture) and V (vaginal culture) on brucella agar: ~ ~ detection o greater than or equal to 1 CFU; 0 c no colonies detected. Controls received nc treatment, Streptomycin sulfate (aqueous) 10 mg/kg body weight, I.P.
c SPLVs containing ~treptomycin 6ulfate 10 ~g/kg body weight, I.P.
~ g -5.1 TP,E~LE XII
RISSULTS O~ CULTI~RES i~ROM ~I55UE 5AMPLES
5~[N B . C~l IS IN~ECTEI~ DOGS SUBJECTED
TO A ~O ~RE ATI~;EN T 2~ T I B ~ 0~ I C RE G LQ~N
~PLY~
b Containing c d e Tissue Streptomycin S~reptomycirl Contrvl Whole blQod 9 Vag:Lnal swab 0 Lun~3 s o + ~
Spleen o +
5yn~3vial f lui~ N. 1). 0 0 Ute r u~ 0 Ovary 0 + +
Pop:L i teal lymph node N . D ,, + +
20 Sal;ivary gland o ~ 0 ~onlsil O +
Med.ias tinal lymph node 0 2i . D .
Mesenter ic lymph node . N . D. 0 25 B~,n,e m~lrrow Superficial N.D. N.l).
cer vical lymph node Axillary lymph ns:~de 0 a Animals treated on day 7 and l0 post-infection.
Samples taken ~t necropsy were serially diluted on brucella agar; ~ ~ equal ts:~ or gr~ater than 1 CFU; 0 ~ no colonies.
c 5PLVs containing streptomycin ~ul~ate, l0 mg/kg body weiyht, I.P.
~ Strep'comycir3 ~ulfate 5aqueous) 9 10 mg/kg body wei9ht, I .P.
e Gontrols received no ltreatment.
-52~
~esult~ of culture ~nd ~erologic ~es~s of dogs infected with B. c~nis before, ~urin~, and after two-stage antibiotic adminis~ration are pre~en~ed in Table XI. All animal~ were serologically negative for pre~iou~ exposure to B. canis as measured by neg~ti~e serum t~ter~, ~n~ were ~ulture negative from blood cultures ~nd cultures of vaginal swabbingsO ~11 animals were noted to be culture po~itive for bo~h blood and vaginal cultures prior to treatments on days 7 and 10. Dogs treated with aqueous 10 streptomycin or dogs receiving no treatment remained 2 culture positive for blood and vaginal cultures during post-treatment periods prior to termination on day 21.
Group 3, which received liposomes containing ~reptomycin, became cul~ure negative one day ~f~er the first treatment 15 and remained ne~ative throughout post~trea~ment period~
Dogs which r~ceived no treatment or aqueou~ ~treptsmycin developed detectable serum titers against B. canis antigens by day 21 post-inoculation, while those treated with SPLVs containing antibiotics on days 7 and 10 20 post-incculation did not develop any detec~able an~ibody to ~. canls antigenO
Results from isolation of B. canis from infected d~gs treated with two-stage antibiotic administration 25 which are pres~nted in ~able XII demons rate that in dogs, only treatment with SPLVs containing ~treptomycin was effective in eliminating any viable B. canis in all tissues from all organ samples.
8.6. TREATMEN~ OF B, ABORTUS IN GUINEA PIGS
Fifteen adult female guinea pigs were inoculated with B. abortus A~CC 23451 (1 x 10 CFU, ~.P.). 5even days post-inoculation animals were divided into 3 groups 35 of 5 animals each~ Gro~p 1, designated Controls, received 7'7 o53-no treatmen~. ~roup 2 r~ eived ~gueou~ ~treptomycin~ulfa~e, I.P., injection~ (0.2 ~) at l0 mg/kg bs~dy weight on day 7 and l0 po~ oclalation wi~ch B, ~bor~s. Group 3 received ;PLV~ containin~ ~treptomycin ~ulf~te I . P.
5 injections ~0.2 mQ) ~t l0 ~g/kg bs~dv weight on ~ay~ 7 and 10 post-inoculation with B ~bortl~. On day l4 pc~st-inoculation with :3. abortus, ~ll animals were ~acr ~f iced and ~pleens were removed, a~eptis:ally homogenized and serially diluted onto brucella agar for 10 îsolation of ~. abortus. Results of ~urviving Bo abortus per spleen ~f ter 4 days incubation, ~re shown in ~IG. 7 Only SPLVs containing ~treptomycin were effective in eliminating B. abortus residing within guinea pig ~pleen.
In ~nimals receiving aqueous streptomycin or no treatment, 15 viable B. 2Ibortus bacteria were be identified.
B . 7 . T~ATMENT OF B . ABORTUS INFECTIO~ IN COWS
Nine heavily infec~ed animals were utilized in 20 this experiment. B. abortus bacterial isolations from -milk and vaginal swabbings became and remained negative for six weeks following treatment with SPLVs containing streptomycin. When infection reoccurred in these animals, bacterial isolations were found only in quadrants of the 25 udder which were positive prior to trea~ment.
Nine cross-bred (hereford- jersey-Brangus), 22-month old, non-gravid, confirmed B. abortus culture-positive cowc were used. At least 4 mon~chs prior 30 to ~he ini~iation of the study, ~he animals were experimentally challenged ~ conjunctivum with 1 x 107 CFU o~ bor~us Strain ~3G8 during mid~gestation, which resulted in abortion and/or B, abortus culture po~itive lacteal or uterine secretions and/or fetal tis~ues.
7~
-5~~
C~ws were maintained in ~ndividual i601ation ~talls and ~epara~ed into three groups. ~reatment comprised a two-dose regimen, ~paced 3 ~ays ~p~rt, as follows~ 3 cows were injected in~raper$toneally wi~h 5 physiological ~aline~ (2~ 3 60W5 ~ere injec~d intraperitoneally with aqueous antibiotic (~Sreptomycin at 10 mg/kg body weight) plus preformed buf~er~illed SPLVs~
(3) 3 cows were in~ected intraperitoneally with SPLV-entrapped streptomycin (10 mg/kg body weigh~). The 10 total volume per injection was 100 mQ per animal.
During the firs~ 2 ~onths duplicate bacteriologic cultures of lacteal and uterine secretions were performed weekly providing secretions were obtainable. Then, all 15 cows were euthanatized with an overdo~e of sodium pent~barbitol, and the following organs were ~ollected in duplicate for bacteriologic cultures~ lymph nodes:
left and right atlantal, left and righ~ suprapharyngeal, left and right mandibular, left and right parotid, left 20 and right prescapular, le~t and righ~ prefemoral, left and righ~ axillary, left and righ~ popliteal, left and right intexnal iliac, left and right supramammary, le~t and right renal, bronchial/ ~ediastinal~ mesenteric, ~nd hepatic, (2) glands: all four quarters of mammary gland, 25 left and ri~ht adrenal glands and thymus (if present);
(3) organs and other ~issuesO spleen, liver, left and right horn of ~terus, cervix, vagina, kidney and tonsil.
After necropsy, all tissues were frozen and 30 maintained at -70C whi~e in transport. Tissues were thawed, alcohol flamed, and aseptically tlimmed prior to weighing. Once weight~ wère recorded (0.2 ~o 1.0 grams), the tissue was homogenized in 1 m~ of 6terile ~aline and serially diluted with steril~ saline to 1:10 10 of 35 initial homogenate 6uspension. Aliquot5 (20 ~Q) of each ~5~-dilutio~ from ~eria~ ~uspensions were pla~ed onto brucella agar and pla~e~ in 37C incuba~:lon. Duplicate de~ermi~a~ions were performed 3or ¢ach ti~8ue.
Plates were read dally and ~cored for bacterial yr owth . All colon ies ~ppear ing pr ior to 3 tiay~ w~re i~ola'ced, pa~aged, and gram 6tained to d~termine iden'city. On day~ 5, 6 ~nd 7 during incuba~ion colo~ies with morphology, grswth, and gram staining characteristics 10 consistent with B abortus were counted; the CF~ per gram tissue was then determined. Representative colonies ~ere repas~a~ed for bacterial confirmation of B. abortus.
Bacteriologic isolations were done on all tissue 5 samples and quantitation of bacteria per gram of tizsue were calculated. The re~ults ~rom four ~nimal --one placebo control and three animals treated with SPLV-entrapped ~treptomycin--are presented in Table XIII.
~ 9. EXAMPLE: TREATMENT O~ OCULAR A~LICTIONS
Bacteri~l and like infections as well as many other afflictions of the eye sause worldwide economic and public health problems, leading, if untreated or improperly ~reated, to los5 of .~ight and possible ~eath due to ~epticemia. Bac~erial infeotions of the eye in animals and man have been reported to be caused by a variety of bacteria including but not limited to:
Clostridiu~ spp., Corynebacterium spp., Leptospira spp., 30 Moraxella ~ Mycobacterium spp., Neisseria p~, Propionibacterium ~e~, Proteus s~, Pseudomonas ~2~t Serratia ~ ~, E. Coli ~ , Staphylo~occus ~
Streptococcus ~p~ and ~acteria~like organisms including Mycoplasma ~ and Ricket~sia ~p. Both ~nimals and man 35 ~erve as reservoirs for potential ~pread o~ ~n~ctious bacteria to each other.
~5 -TABLE X I I I
RES~JLTS OF Ct)LTl~RE,S I~RO~5 TISSUE
SAM PLE S OF E~ . ABORI~US IN P`ECTED Co~a S
l~ntreated SPLV-Entrapped ~;tr@ptomycin Ti sgue Corl tr ol 1 ~ 3 Adrenal gland I. 0 0 0 O
Ad~erlal gland R ++ o O
Atlantal I~ R ++ ~ o Atlantal LN L 0 0 5 +
10 P.xillary LN R ~+ 0 ~ O
Axillary LN L ~+ 0 ~ O
Br onchial T!N 0 0 0 0 Cervix 0 0 0 0 E~epa tic LN ~++~ 0 0 ~orn of Uterus L 0 0 0 +
Horn of U'ceru~ P< 0 0 0 O
Int. Illiac LN R ~+ 0 0 O
~n~. Illiac LN L t~ 0 ~ O
Kidney 0 0 0 0 Liver 0 0 0 0 Lung 0 0 0 0 Mammary Gland LF 0 + + O
Mammary ~land LR 0 0 0 Mammary Gland RF ~ o ~ o 20 Mammary Gland RR +~ 0 0 0 Mandibular LN R +~+ 0 û O
~andibular LN L ~++ 0 0 0 Mediastinal LN ~+ 0 + 0 Mesenter ic LN ++~ 0 0 0 Parotid LN L ~+ 0 0 O
Parotid LN R ~ 0 0 Popliteal LN L ~ 0 0 0 25 Popliteal LN R + 0 0 Prefemoral LN IJ ~ 0 0 O
Prefemoral I,N R û 0 0 O
Prescapular LN L 0 0 0 Prescapular LN R +~ 0 0 0 Renal LN 0 0 0 Spleen +++ 0 0 0 Supr amasNnary LN L t~ ~ 0 0 SupramamlDary LN R 0 0 a o Suprapharangeal LN L ~ 0 0 0 Suprapharangeal LN R 0 0 0 0 Thymus 0 0 0 0 Vagina ~ 0 0 0 ~5 0 No de~e~table b~cteria by cl~lture ~f 0. 3 - 1 gm of tissue .
Less than 200 colonie~/gm tissue.
More than 3û0 c~lonies/gm~
More than 1, 000 colorlie~/gm ~
~+~ More than l0û,Q00 colonie /gmO
~a~6~r~
Such bacterial infection cannot be trea~ed with antibiotics without lengthy and cumb~r~me tr~atment ~chedules re~ulting in either ~requent treatments, as rapid as every twenty minu~e~ in humans w~h ~me 5 infections~ ~r unacceptably high concentration~ ~ the antibiotic in the tissues7 Curren~ ~reatment ~ethods are difficult for many other reasons. The infectiou~ organism in the ~urface tiss~es of the eye in ~ome cases ~re highly resistant to bactericidal activities of antibiotics, and 10 topical a~ministration o~ antibiotic~ can result in rapid clearing of the drug from the eye ~ocket yiel~ing varying contact times. As a general rule, treatment of eye infection6 has to be completely effective ~ince any remainin~ infection will simply reinfect through lacrimal 15 gecretions and the cycle commences once again. ~urther, in many cases drug concentrations needed to eliminate the infection can cause impairment of ~ision and in certain cases can result in total blindness. The economic impact of such diseases in domestic animals is demonstrated by 20 the millions of dollars which are lost each year since the only potential way to combat ~uch infectious diseases is sustained therapy and quarantine.
The following experiments evaluate the 25 effectiYeness of treatments using free ~ntibiotic in glycerine as csmpared to antibiotic entrapped in SPLVs for M. bovis infections of the eye~
M. bovis causes infectious keratocc>njunctivitis __ 30 ~pink-eye) in cattle~. This condition is characterized by blepharospasm, lacrimation, conjunc~civitis and varying degrees of corneal opacity and ulceration. Adult cows may develop a mild fever with sligh~cly decreased appetite and a decreased milk pr~ductionc Although a nu~ber of 35 antibiotics are effective again~t M~ bovls, they must be ~dmini~tered early and repeated o~t~n by topical application or ~uboonju~tival ~nject~onO
According to the example~ de cribed herein, the 5 e~fectiveness and duration of action o~ the therapeutic ~ubstance are prolon~ed. I~ i6 ~urpri~ing that this ~ytem ix effective with only one or two adminis~rations ~lnce such infections do not re~pond to simple ordinary treatment with antibiotics. The usual treatments often 10 leave small remaining in~ections which reinfect the eye so that ~he infectious cycle will commence again~ unless the infection is completely eradicated by ~umerous repetitions of the treatment.
9.1. TREAIMEN~ OF IN~ECTIOUS KERATOCONJUNCTIVITIS IN MICE
C57 black mice (160 mice~ were divided into groups. One half ~f each ~r~up was exposed ~o ~.V.
irradiation in each eye (in order to create corneal 20 lesions). All animals were then inoculated with M. bovis instilled onto the right eye at concentrations of 1 x 106 bacteria per eye. Twenty-four hours post-inoculati~n all animals ~ere scored for degree of corneal opacity. The eight groups were treatea by topical 25 application of the following to each eye: Groups 1 and 2 received 10 ~Q of SPLV-entrapped ~treptomycin (30 mg/m~);
Groups 3 and 4 received 10 ~Q streptomycin (100 mg/mQ);
Gro~ps 5 and ~ received 10 ~Q of buffer-fil1ed SPLVs ~uspended in aqueous streptomycin (100 mg/mQ); and Groups 3~ 7 and 8 received 10 ~Q of sterile ~aline (N.B. The uninfected left eyes were treated with the same topical solutions in order to determine whether SPLVs would irritate the eye; no irritation was observed). Once daily, animals were scored for progre~sion or regression 35 of corneal lesions ~nd vn days 3, 5 and 7 post-treatment -5g-right eyes were ~wabbed and i~41.~ion8 for MD bovi~ were performed on repre~entative animals~ M. bo~ olonies were de~ermined by colony ~orphology and reac~i~lty to flour@6cently l~beled antibvdy 1:o M. ~ovi~ pil~ J, Re~ults, ~h~wn in Table XIV, re~eal that o~ly the SPLV~entrapped treptomycin was effective in elimina~iny infec'cion..
902~ TREATMENT 0~ RABBIT CONJUNCTIV~
~SING SPLV~ENTRAPPED ANTIBI8TIC
pl~. bovis, ~TCC ~rain 10900, were diluted to a concentration of 1 x 107 cell~ per mQ in ~terile aline t0.08596 NaCl). Aliquots (0.1 mQ) o~ bacterial ~uspensions were inoculated ~opically into the eyes of ten ~dult 15 ~emale rabbit~. Samples ~or cultures were taken daily by 6wabbing the conjunctivae and pla~ed onto blood agar plates ~or isolation of M bo~ls. Three days post-inoculation, rabbits were divided into 3 groups: 2 animals (controls) received no treatment; 4 animals 20 received treptomycin in ~terile saline (concentration lO
mg/kg body weight); and 4 animals received SPL~-entrapped streptomyein in a ~terile saline solution (concentration lO mg streptomycin/kg body weight). ~ll solu~ions were administered 'copically into each eye . Af ter 24 hours, the 25 swabbing of conjunctivae of all rabbits wa~ resumed and continued daily ~or seven daysO The results of isolation for M bovis ~n blood ~gar plates are shown in Table XY.
~ , .
9.3. TREAlMENT 0~ RERATOCONJUNCTIVITIS
~ESULTING ~ROM SUBCUTANEOUS INFECTIONS
M. bovis, ATCC ~train lO900, were diluted to a _ _ _ _ . _ c:oncentration of 1 x 107 cell~ per mQ in l;terile saline~ Aliquots (O.l mQ) of bac~erial su~pensions were 35 inoculated into the eyes of adul~ rabbit6 which had been TABLE XIV
~E SULTS OF TREATMEN ~ OF Ii~d E'E CT IOU
KERATOCONJ[~NCTIVITIS R~S17LTING FiRC~
OCULARs INFECTIC)N~ OF M . BOVIS IN MICi3 M~ Bovis Number of Mice Per Group of 20 Culturesa Pre-Treatmen~ Post-Tr~a'cment Days Post-Corn~al Qpacityb Corneal Opacityb Treatment D l 2 3 4 0 l 2 3 4 3 5 Non-radiated Mice Controls 16 3 0 l 0 18 2 0 0 04/5 4/5 I?r ee S~reptomycinC 18 1 1 0 O lB 2 0 O 02/5 ~/5 BLlf r e r -f i 1led SPLVs pl u s f r ee Streptomycin~ 17 2 l 0 0 18 l l 0 02/j 3/5 SPLV~-En tr apped StreptomycinC 17 3 0 0 0 20 0 0 0 00/5 0/5 W-Radi~ted Mice Controls l l 5 9 4 l0 3 l 2 45/5 5/5 Fr ee StreptomycillC O 4 9 7 0 14 ~ 2 1 03/5 4f5 Buffer~filled SPLVs plus free StreptomycinC 0 3 5l0 2 ll 2 4 3 03/5 3~5 SPLVs - En tr apped StreptomyGinC 0 l 5ll 3 19 l 0 0 00/5 0/5 a Culture c>f eyes positive f~r presence of ~3. bovis, determined by ~luorescent antibody staining.
b Scoring of norlDal cornea: 1 8 loss of normll luster;
~ - small foci o~ opacity; 3 ~ partial opacity Of cornea; 4 = total opacity of cornea.
Total administration l0 llQ ~l,0 mg streptomycin per ~Y~ ) .
~ABLE XV
5RE5ULTS OF ISOL~ION ~OM RAB~IT
CO~JtJNCl~lVAE ~TER ~OPICALLY IN~ECTING
WTTH M BOVIS AND TREATING ~ITH
AQUEOUS OR~PLV-ENCAPSULATED STREPTOMYCIN
M. bo~ Culture~a An imal D~y s P~ t - I n ~ec t i on Group Number Pre-Treatmen-b Post-~reatmentC
3 ~ 5 6 7 Con tr ol l 0 ~ + ~ ~ + +
2 0 ~ ~ + ~ ~ +
Str eptomycind l 0 ~ ~ +
2 0 0 ~ ~ + +
3 ~ ~ ~ +
4 0 ~ + + + ~ +
SPLY Entrapped 1 0 0 ~ O 0 0 O
Str eptomycine 2 0 + ~ 0 0 0 0 3 0 + ~ 0 0 0 4 û + + O 0 0 O
a Cultures scored for presence of M. bovi~ colonies on blood agar plates after 24 hours at 37C. Plus (~) represents greater than or equal to 1 CFU ~3. bovis per i~olate; 0 represent~ no detectable colonies.
25 b All animals inoculated with l x l0S CFU M. bovis topically in each eye.
c Animals ~reated with 0 O l m~ solution topically in each eye~
d Streptomycin (l0 mg/kg body weight) in sterile saline ~olution.
SPLV-entrapped ~treptomycin (l0 mg/kg body weight) in ~terile saline ~olu'Lion.
--6;~o pr~viou~ly infected a~ de~cribec9 in Section 9. 2. ~nd were not treated with SPLVs. The ri~ht eye6 of all nine rabbits were irloculated wi~h 0.:L mQ of M. bovis ~ubcutaneou~ly into conjunctiva:L ti~ue and in the left 5 eyes of ~ll rabbits s~/ere irloculated wi th 0.l m~ of M.
bovis ~opically. Cultures we~e taken daily from __ 6:onjuncti~ae of both eyes from all rabbits And plated onto blood agar plates for isolation of Mo bovis. Three days post-inoculation, rabbits were divi~ed in~o 3 group~: 2 0 animals received no treatment; 3 animals received streptomycin in a ~tandard ophthalmic glycerin ~uspension (concentration of streptomycin lOmg~kg body weight~; ~nd 4 ani~al~ received a 6aline 6u~pension of SPLV-entrapped streptomycin tlO mg of streptomycin ~ulfate per kg of body 15 weight). The.~uspension or 501UtiOTI was a~mini~tered topically (0.l mQ) into each eye. After 24 hour~ ~nd on each o~ the next five days, conjunctiYal swabbings were taken from all rabbits. The results of isolation for M.
bovis on blo~d agar plates are shown in Table XVI.
20 Necropsies were performed on all animals at the termination of experiments and conjunctivae were removed from all animals. These were ~cored for vascularization, and were minced, hom~genized and plated onto blood agar plates for isola~ion of M. bovis. Results are shown in 25 Table XVII.
9 . 4 . EVAL~ATION OF THE EFFECTIVENESS OF SPLVS
AS COMPARED I~O LIPOSOME PREPARATIONS IN
T~: TREATMEMT OF OCULAR INFECTIONS
M. bovis (ATCC strain 10900) were diluted to a concentration of l x l0 ' cells per mQ in 6terile ~aline. Aliquo'cs IOol mQ) of bacterial su~;pensions were inc>culated subcutaneously into the conjunctival tissues of 35 both eyes in adult rabbi~s. Swabbings were ~aken daily 7~
~63-~ABLE XVI
RESI~LTS 0~ ISOLA~ION P`P~OM RP~BBIT CONJUI~Cq`IVAE
AFTER INVCVLA~ION OF Pq ~ BOVIS INTO
~NJUNC~IYAL ~BRANES ~3D TREATMENI g~TH
ST~PTOMYCIN IN OPH~ALMïC GLYOERINE S9I-UTIC
OR SPLV-ENCAPSIJLATE:D STREPTOP~YCIN IN SALINE
P~. bo~i~ Culturesa Day~ Po~ t Infec t i on An imal Group Numberb Pre-treatment Post-Treatment Corl tr ol 1 +
~trep~omyc in l ~ +
in Glycer ine 2 +
solutlon d 3 + ~ ~ +
SPLV~ û O O
Encapsulated 2 ~ + O 0 O
20 Str eptomyc ine 3 + + 0 0 0 ~ 4 ~ ~ ~ 0 0 a Cultures scored for presence of M. bovis colonies on blood agar plates af'cer 24 hour~ ~t 37~C. Plus ~+) represents greater than or equal to 1 CFU M. bovis per i~olate; 0 represents no detectable co~nies.
b All animals were inoculated with 1 x l06 CFU M.
bovis topically in both eyes; l x lO6 CFU M. bovis was lnjected into conjunctival membranes, ln right eyes; and l x lO6 CFU M. bovis was applied topically in le~t eyes.
3o c Animals treated with 0. l mQ solution tc~pically in each eye.
d ~imals treated topically in ~ach eye with 6treptomycin (lO mg/kg body weight~ in t)ph~chalmiC
glycer ine base ., 35 e Ar imals ~reated ~opica:lly in each eye wi~h SPI.V-encapsulaed ~treptomycin ~lO mg~kg bc~dy weight) in sterile ~aline 60lution.
6~7 ~6~-TABLE XVII
RESULTS ~ROM N~CROPSY OF T}~ ORBIT AND AS~OCIATED
TISSUE~ FROM R~BBITS ~FTER ~M~LA~ION WIT~ M~ OVIS
INTO CONJUNCTIYAL TISS~ES ~ND ~EATMENT WIT~ EI~HER
STREPTOMYCIN IN OP~THALMIC ~LY OE RINE SOLU~IOM ~R
SPLV- NCAPS~LATED STREPTOMYCIN I~ STERLINE SALINEa Isol~tion of M. bovis Vascularization Cultures of Right Eye b Control A ~ 2+
B + 2 Streptomycin in 15 ~lycerine Sol~tion A ~ 2+
B + l~
C ~ 2+
D + 2+
20 SPLV-encapsulated S~reptomycin O O
~5 a Legends are same as ~able XII, performed on day 5, post infection.
b Vascularization scored as follows: 0 = vessels normal; l = some vessels definitely dilated and infiltrated by minor vessels; 2 = diffuse red with individual vessels not easily discernible; 3 =
diffuse bee~y red, vascular leakage and effusion of blood into conjunctivae~
-65~
~rom conjunctivae of both eye~ from all rabbits and plated onto blood agar plate~ for i~olation of M~ bovi~. ~ive day~ po~t-inoculation, rabbits were divi~d ln~o 6 groups: 2 animal~ received no tre~tment (contro-l~); 3 - ~ anim~ls ~eceived a ~uspension of SP~V-encapE~la~ed streptomycin (lOmg of s~reptomycin ~ulfa~e per kg of body weight) which when diluted 1~100 had an O~D.4~0 (optical density at 480 nm) equal to 0.928; 3 animals received a suspension of SPLV-encapsulated ~treptomycin (10 mg of ~treptomycin ~ulfa~e per kg of body weight) which when dilu~ed 1:100 had an O~D~480 equal to 0.449; 3 animals received a ~uspension of SPLV encapsulated strep~omycin (10 mg streptomycin ~ulfate per kg of body weight) whi~h when diluted l:lOG had an O.D.480 equal to 0.242; 3 animal~ received a 6~spension of SPLV-encap~ulated ~treptomycin (10 mg 6treptomycin 6ulfate per kg body weight) which when diluted 1:100 had an O.D~80 equal ~o 0.119; and 2 animals received a suspension of multilamellar vesicles (MLVs) containing streptomycin (10 20 mg strep~omycin ~ulfate per kg of body weight) wi~h an O.D.480 of a 1:100 dilution equal to 0~940. MLVS were made ~y the process of ~ountain et al. Curr. Micro. 6:373 ~1981) by adding ~treptomycin ~ulfate to the dried lipid film which was then vortexed, and allowed to swell for two 25 hour s; ~che non-entrapped streptomycin was removed by repeated centr if ~gation.
The suspensions were administered topically into each eye. After 24 hours, conjunctival swabbings were 30 taken from all rabbit~ àaily for g days and plated onto blood agar. The results of isolation for M. bovis on blood agar plates ~re shown in T3ble XVIIIo Necropsies were performed on all animals. These were scored for lacrimal secretions, anà conjunctivae were removed 35 aseptically ~Erom all animals. These were scored for 8~7~9 ~66-~a~ularization~ and were minced, homogen~zed ~nd pl~ted onto blood ~gar plate~ for i~olation of M is~ Result are shown in Table XIX.
10. EXAMPLE: TR~:A~ENT O~ VIRAL IN~ECTIONS
Lymphocytic choriomeningitis viru~ (LCMV)~ ~
member of the Arenaviru~ group, is known to cau~e diseases in man and LCMV infection is fatal in mice inosulated 10 intracerebrally with this virus. The death of mice is caused by the immune cells which react against virus-infected cells. The virus does not kill the cells in which it ~ltiplies, therefore, the therapeu~i~ agent used in mice mu~t either inhibit ~irus multiplica'cion ~o 15 tha~c the inunune cells will not be act~Yati?d, ~nd/~r inhibit the actiYation of immune cell~.
The following example demonstrates the effectiveness of trea~ing viral infections by 20 administering a SPLV-encapsulated antiviral compound.
10.1. ~REATMENT O~ LETHAL LYMPHC~Y~IC CHORIO-~SENINGITIS VIRUS INFECTIONS IN MICE
5wiss mice 2 months of age were inoculated intracerebr~lly with a lethal dose of LCM virus, i ~., lO0 plaq~e forming units (P~U) in 0.05 mQ inoculum per mouse.
Mice were divided into 4 groups of 7 animals each and were ~reated on days 2, 3 and 4 pos~-infection by 30 in~raperitoneal injections with O.l mQ/dose/mouse as follows~ he ~5PLV-R group" was treated with a ~uspension of e99 phs:~sphatidylcholine SPLVs contalning 3 mg Ribavarin/m~. SPLVs were prepared using 100 mg lipids and 0.3 mQ of lO0 mg drug/mQ in PBS buffer; ~he ~ntrapment ~67~
TABLE XVIII
ISOLATION OF M. BOVIS FROM INFEC~ED RABBIT
CONJUNC~IYAE A~TER ~REATMENT ~fT~ DILUTIO~S
0~ SPLV-ENCAPSULATED STREPTOMYCIN I~ ~ALINE
OR MLV-ENCA~S~LATED STREP~OMYCIN IN SALINE
I,olation sf ~. b~v s ~ay~ Po~t~In ect.ol Animal Pre-Treatmen. Post~ eatment Group Number 1 ~ 3 4 6 7 8 9 ....... 11 12 13 14 Co~ol 1 + ~ ~ + ~ + + ~ + +
2 + ~ + + + ~ ~ + + +
MLV-15 encapsulated 1 + ~ + ~ 0 0 + + +
Streptomycin 2 t ~ O ~ ~ O O + ~ O
SPLV-encapsulated 1 + + ~ ~ + 0 0 0 0 0 0 0 0 0 Streptomycin 2 + ~ ~ ~ + 0 0 0 0 0 0 0 0 0 (undiluted) 3 ~ ~ ~ + + 0 0 0 0 0 0 0 0 SPLV-encapsulate~ 1 ~ + + + + ~ 0 + ~ ~ + +
Streptomycln 2 + + ~ ~ ~ 0 D + 0 0 0 0 0 0 (1:2 dilution) 3 ~ ~ + ~ + 0 0 0 0 ~ 0 0 0 0 SPLV-encapsulated 1 + ~ + ~ + + + 0 0 0 0 0 0 0 25 ~treptomycin 2 + + ~ + ~ ~ 0 0 0 + 0 0 0 0 (1:4 dilution) 3 ~ ~ + + + 0 0 + ~ ~ + ~ ~ +
SPLV-encapsulated 1 + ~ ~ ~ + 3 0 ~ + + + ~ ~ +
Strepto~ycin 2 + ~ 0 0 0 0 0 0 0 0 0 ~1~6 dilution) 3 ~ + ~ + + 0 + ~ + ~ +
3~
a All animals inoculated wi~h 1 x 106 C~ M. bovis by injecti~n into conjunctival membranes o~ both eyes.
Conjunctival swabbings were plated on ~l~od agar.
Cultuzes scored for presence of M. ovis colonies on blood agar plates after ~ hours a~ 37~C; + ~ greater than or equal to 1 CFU; 0 = no detect~ble cultures.
TABLE XIX
~ESULT5 FROM NECRO~SY OF ~E~ ORBIT A~D
A~SOCIATED TISSUES FR0M RABBI~S AFTEP~
INOCULATION WIT~ M. BOVIS IN~O CONJUNCTIY~L
~ISSUES AND TREATMEN~-~IT~ EI~HER MLV-ENCAPSULATED
STREP~OMYCIN, SPLV~ENCAPS~LATED STREPTOMYCIN
OR DILVTION~ OF SPLV-ENCAPS~LATED STREPTOMYCINa Isolation Vasculariza~ Lacrimal Animal of M. Bovis tion o~ Eyesb DischargeC
Control l + l+ l+
2 ~ l~ l+
MLV
encapsulated l + l+ l+
15 S~rep~omycin . 2 0 l+ 0 encapsulated l 0 0 0 Strep~omycin 2 0 l~ 0 (~ndiluted) 3 0 0 0 SPLV-20 encapsulated l ~ 2+ 2+
Streptomycin 2 0 0 0 (l:2 dilution) 3 0 l+ 0 SPLV-encapsulated l 0 0 0 Streptomycin 2 0 l~ 0 25 (l:4 dilution) 3 + l+ 0 SPLV-encaps~la~ed l + l~ l+
Streptomycin 2 0 l~ 0 (1:6 dilution) 3 ~ l~ 0 3o a Legends are same as Table XIV, per~ormed on day 14 post-infection.
b Vascularization scored as follows: 0 = ~essels normal, l ~ome vessels dilated and infiltrated by minor vessel~; ~ = diffuse red with individual vessels not easily discernable; 3 - diffuse beefy r~d/ vascular leakage and e~f~sion of blood into conjunctivae.
Discharge ~cored as follows: 0 c no discharge; l =
di~charge with moi~tening of lids ~nd hairs adjacent to lids0 2 = discharge with moistening of lids, hairs ~nd areas ad~acent to eyes.
7~
TABLE XX
6 T~EATMENT O~ LETHAL LCM VIRUS IN~ECTI~W IN ~ICEa Gr oupLe'Lhali~cyCVirus P~ecovered from Splee (PYU x 105~ Q) Control 5/5 7. 0 SPLV-gr oup 5/5 6 ., 9 ~R group 5/5 5O 2 SPLV-R Gr oup 3/5 3 ~ 4 Two month old mice were each inoculated intracerebrally with a lethal dose~ lO0 PFU of LCM viru~ in 0.05 mQ inocula.
b Lethality is expressed as number dead/number in group.
c On the fi~th day post~infection 2 mice from each group were sacrificed and their spleens homogenized at a concentration o~ l gm ~pleen/20 mQ homogenate.
of drug was 10~; (2) the ~R-groupe' was treated with a ~olution of Ribavarin 3 mg/m~ in PE~S; (3) the "SPLV-~roup"
25 was ~rea~ed wi~h buffer~filled 5PLVs (i.e~, SPLVs prepared a~ above bu~ without Ribavarin); and ~4) the "control group" was tre~ted with PBS. On day 5 post-infection mice from each group were sacrificed and their spleens homogen i zed ( 2 6pleen s~gr oup wer e homog2n i zed in PBS a t 3~ 1/20 weight per volume buffer). 'rhe plaque ~orming units (PFU~ per mQ were determined fs?r each suspension~ The remaining 5 mice in each groups were obserYed for lethality two ~:ime~ daily for 30 day~. The re~;ul'cs ~re presented in Table XX.
~5 --7 ~--~ rable XX clearly indica~e~ a decrease in lethali~y and a ~3ecrease in th~ viru~ recoverable ~rom the ~nfected animal~ We have nst ye~c determined whe~her ~he~e resul~s are due ~co the anti-viral wtlvi~y o~ the 5 ribavarin which i~ relea~ed from the SPLYs i~r whether it i~ due to an i~ununomodulation of the mouse ho~t durin~ the su6tained release of rib~varin ~rom the SPLVs.
Claims (23)
1. Stable plurilamellar vesicles comprising lipid vesicles ranging from about 100 nm to about 10,000 nm in size, characterized by a few to over 100 lipid bilayers enclosing aqueous compartments containing at least one entrapped solute in which the lipid bilayers have an ordered molecular architecture creating a supermolecular structure which differs from that of other multilamellar vesicles so that when compared to other multilamellar vesicles composed of the identical lipid and aqueous ingredients, stable plurilamellar vesicles have the following properties:
(a) a higher percent entrapment of solute;
(b) a lower buoyant density;
(c) a volume about one-third larger;
(d) greater stability to auto-oxidation during storage in buffer;
(e) greater stability in body fluids;
(f) a larger percent leakage of entrapped solute when exposed to urea, guanidine, or ammonium acetate;
(g) a smaller percent leakage of entrapped solute when exposed to hydrochloric acid or serum; and (h) distribution of entrapped contents throughout the cytosol of cells when administered to the cells in culture.
(a) a higher percent entrapment of solute;
(b) a lower buoyant density;
(c) a volume about one-third larger;
(d) greater stability to auto-oxidation during storage in buffer;
(e) greater stability in body fluids;
(f) a larger percent leakage of entrapped solute when exposed to urea, guanidine, or ammonium acetate;
(g) a smaller percent leakage of entrapped solute when exposed to hydrochloric acid or serum; and (h) distribution of entrapped contents throughout the cytosol of cells when administered to the cells in culture.
2. Stable plurilamellar vesicles according to claim 1, wherein said vesicles are substantially free of multilamel-lar vesicles (MLVs), small unilamellar vesicles (SUVs), and reverse-phase evaporation vesicles (REVs).
3. Stable plurilamellar vesicles according to claim 1, wherein the major lipid component of the vesicles is a phosphatidylcholine.
4. Stable plurilamellar vesicles according to claim 1, wherein an anti-oxidant is a component of the vesicle.
5. Stable plurilamellar vesicles according to claim 4 wherein said anti-oxidant is butylated hydroxytoluene.
6. Stable plurilamellar vesicles according to claim 1, wherein a protein is entrapped within the vesicle.
7. Stable plurilamellar vesicles according to claim 1, wherein a compound selected from the group consisting of: antibacterial compounds, antifungal compounds, antiparasitic compounds, and antiviral compounds is entrapped within the vesicle.
8. Stable plurilamellar vesicles according to claim 1, wherein a compound selected from the group consisting of: tumoricidal compounds, toxins, cell receptor binding molecules, and immunoglobulins is entrapped within the vesicle.
9. Stable plurilamellar vesicles according to claim 1, wherein a compound selected from the group consisting of: anti-inflammatory compounds, anti-glaucoma compounds, mydriatic compounds, and local anesthetics is entrapped within the vesicle.
10. Stable plurilamellar vesicles according to claim 1, wherein a compound selected from the group consisting of: enzymes, hormones, neurotransmitters, immunomodulators, nucleotides, and cyclic adenosine monophosphate is entrapped within the vesicle.
11. Stable plurilamellar vesicles according to claim 1, wherein a compound selected from the group consisting of: dyes, fluorescent compounds, radioactive compounds, and radio-opaque compounds is entrapped within the vesicle.
12. A method for preparing stable plurilamellar vesicles, comprising:
(a) forming a dispersion of at least one amphipathic lipid in an organic solvent;
(b) 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 (c) concurrently emulsifying the aqueous phase while evaporating the organic solvent of the biphasic mixtures, wherein the stable plurilamellar vesicles produced are substantially free of MLVs, SUVs, and REVs.
(a) forming a dispersion of at least one amphipathic lipid in an organic solvent;
(b) 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 (c) concurrently emulsifying the aqueous phase while evaporating the organic solvent of the biphasic mixtures, wherein the stable plurilamellar vesicles produced are substantially free of MLVs, SUVs, and REVs.
13. The method according to claim 12, wherein the ratio of volume of solvent to volume of aqueous phase is from about 3:1 to about 100:1.
14. The method according to claim 12, wherein the temperature at which the method is performed is from about 4°C to about 60°C.
15. The method according to claim 12, wherein the temperature at which the method is performed is less than the phase transition temperature of at least one of said lipids.
16. The method according to claim 12 f wherein the solvent is fluorocarbon or diethylether, or mixtures thereof.
17. The method according to claim 16, wherein the solvent contains an anti-oxidant.
18. The method according to claim 17, wherein said anti-oxidant is butylated hydroxytoluene.
19. The method according to claim 12, wherein a material to be entrapped in the vesicles is added with the aqueous phase.
20. The method according to claim 19, wherein at least 20 percent of said material is entrapped in the vesicles.
21. The method according to claim 19, wherein said material is a protein.
22. The stable plurilamellar vesicles of claim 1 in which the lipid bilayers comprise a phospholipid.
23. The stable plurilamellar vesicles of claim 22 in which the phospholipid is zwitterionic.
Applications Claiming Priority (11)
Application Number | Priority Date | Filing Date | Title |
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US36299482A | 1982-03-29 | 1982-03-29 | |
US36299582A | 1982-03-29 | 1982-03-29 | |
US362,995 | 1982-03-29 | ||
US362,994 | 1982-03-29 | ||
US41146682A | 1982-08-25 | 1982-08-25 | |
US411,466 | 1982-08-25 | ||
US44724782A | 1982-12-06 | 1982-12-06 | |
US447,247 | 1982-12-06 | ||
US463,900 | 1983-02-04 | ||
US476,496 | 1983-03-24 | ||
US06/476,496 US4522803A (en) | 1983-02-04 | 1983-03-24 | Stable plurilamellar vesicles, their preparation and use |
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CA1198677A true CA1198677A (en) | 1985-12-31 |
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CA000424498A Expired CA1198677A (en) | 1982-03-29 | 1983-03-25 | Stable plurilamellar vesicles |
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