Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
--3--
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~
-4-
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,
5--
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~
--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
77
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~
- 9 -
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.
-12-
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
13 -
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
15 -
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~
19 -
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
23-
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
--28--
~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
-29-
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 .
30-
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~
-31-
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
-32-
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.
77
-.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
.
-36-
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 .
-410-
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;~
4 3
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
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~
-45-
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
-49-
~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~
-57-
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
0
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
--6U--
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
2 3 4 5
Corl tr ol 1 +
1 5
~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
A 0 0
B 0 0
O O
D 0 0
~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.
-68-
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
5PLV-
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.
