Note: Descriptions are shown in the official language in which they were submitted.
WO9S/12386 2 1 7 ~ 3 2 6 ~ /03668
~u~ n LIPOSONE ~RE~ARA~ION
Back~round of the Invention
This invention relates to liposomes and their
a~;n;~tration to host org~n;~c.
Liposomes have been used as carriers for active
agents, including therapeutic, diagnostic, and
prophylactic agents.
Liposomes generally comprise amphipathic compounds,
having a hydrophilic head and a hydrophobic tail, which
react under certain conditions to form a bimolecular leaf
structure of at least two layers of lipid, in which the
polar head groups are at the interface between the
aqueous medium and the lipid and the hydrophobic tails
interact to form an environment that excludes water. The
lipid bilayers are stable structures held together by the
non-covalent interaction of the hydrocarbon groups of the
acyl groups. When the lipid bilayer closes in on itself,
it forms a spherical vesicle called a liposome having an
internal space separated from the external environment.
Desired agents may be encapsulated in this space, or
within the bilayer, itself.
The lipids which comprise the liposome are typically
phosphoglycerides, such as phosphatidyl choline
(lecithin), and sphingolipids.
Liposomes may be used as carriers for, e.g., thera-
peutic, diagnostic, and prophylactic agents. They are
especially advantageous in that they may protect suscep-
tible agents from degradation, thereby increasing the
time during which the agent is active in the body. In
UrE SHEET ~RVLE 26)
2 1 7~326
WO9S/12386 ~ Sl~3668
-- 2
addition, tissue or cell specific targeting of the lipo-
some may be achieved by incorporating a component into
the liposome, e.g., coupling, conjugating, absorbing or
adsorbing it to the liposome bilayer surface, which is
selective for a specific tissue or cell type. For
example, lipids, antibodies, lectins, receptors, ligands,
and other such components may be incorporated into
liposomes for the purpose of achieving tissue specific
targeting.
A further application of liposome technology is in
the treatment, prophylaxis, and diagnosis of liver
disorders. In spite of the large variety of imaging
techniques and modalities currently available, accurate
assessment of liver lesions remains a difficult
diagnostic problem. One of the reasons for the
diagnostic difficulties is the low contrast difference
between normal and tumor liver parenchyma, which only
allows delineation of lesions above l to 2 cm.
Accordingly, it would be desirable to be able to
concentrate a radiographic or MRI contrast medium in the
liver in order to increase the contrast for a time period
that permits thorough ~YA~; nAtion of the entire organ.
One way to achieve uptake of hydrophilic substances
into the liver is to encapsulate them in liposomes.
After i.v. administration, liposomes may be taken up by
the phagocytic cells of the reticulo-endothelial system.
Thus, they are chiefly concentrated in the Kupffer-cells
of the liver and macrophages in the spleen. Since
phagocytic activity is not displayed by tumor tissue, the
liposomal contrast agent can be concentrated selectively
in the healthy tissue. This results in an increased
density difference (~ HU for X-ray imaging, ATl or ~T2
for MRI).
Various investigators have been able to demonstrate
the effectiveness of such an approach in principle. In
most cases, however, they have been unable to achieve a
SIJB~ITU~ SHEET fRULE ~6~
WO9SI~2386 2 1 7 4 3 2 6 PCT~4/03668
- 3 -
sufficient density difference due to the low iodine-
loading capacity of their liposomes; nor have any of the
methods of preparation for the contrast agent carrying
liposomes applied proven suitable for reproducible large-
scale production of pharmaceutically acceptable liposomepreparations (adequate shelf-life, sterile, pyrogen free,
high iodine encapsulation).
A method of preparation of contrast-carrying lipo-
somes has been developed which overcomes all the above
limitations. See WO 91/16039; P 40 135802. This process
called "ethanol-evaporation method" enables reproducible
production of large amounts of liposomes under aseptic
conditions. The original liposome suspension produced by
this method is lyophilized to obtain a product with an
adequate shelf life. Prior to application, the lyophili-
zate is resuspended with 135 mM mannitol solution to give
a liposomal suspension which is infused after a
filtration step.
Using contrast-carrying liposomes produced by this
method, opacification of the liver and spleen has been
achieved. On the basis of initial biodistribution
studies, iopromide was chosen as liposomal contrast agent
because its 7-day retention values in the liver and
spleen are significantly lower than those of iotrolan.
Although liposomes are potentially an important
advance in drug delivery systems, such as for carrying
contrast media as described above, their use in living
org~n;s~C is limited owing to adverse systemic side
effects which may be observed upon liposome
administration. These systemic effects include, e.g.,
transient hemodynamic depression, affecting blood
pressure, heart rate, and ECG; depression of blood
counts. For example, it is ~nown that liposomes may
have adverse hemodynamic effects when administered
intravenously to animals, posing serious hazards to the
safety of the host animal.
~ rn~ SHEET~R~LE2
WO9S112386 2 1 7 4 ~ 2 6 ~ 3~103668 ~
In view of the occurrence of such systemic effects,
it may not be possible to use such liposome preparations
for drug delivery. Surprisingly, it has been found that
when these same liposomes are prepared but incorporating
an electrically-charged component the adverse systemic
effects upon administration are reduced or eliminated.
Brief DescriPtion of the Drawinq
Figure l is a schematic diagram of the ethanol-
evaporation method of making liposomes.
Figure 2 is mean blood pressure (percent of
prevalue) following intravenous infusion of liposomes
made from different lipids (300 mg lipid/kg, lO0 mg
lipid/kg/min) and mannitol (300mM), n=6, means+SEM.
Mannitol o DSPC/DSPG O DSPC
Figure 3 is total peripheral resistance (percent of
prevalue) following intravenous infusion of liposomes
made from different lipids (300 mg lipid/kg, lO0 mg
lipid/kg/min) and mannitol (300mM), n=6, means+SEM.
Mannitol o DSPC/DSPG O DSPC
Figure 4 is enddiastolic pressure (EDP) following
intravenbus infusion of liposomes made from different
lipids (300 mg lipid/kg, lO0 mg lipid/kg/min) and
mannitol (300mM), nS6, means+SEM.
Mannitol o DSPC/DSPG O DSPC
SummarY of the Invention
By incorporating an electrically charged component
into liposomes which have an adverse systemic effect, it
has be~n found that the resulting liposomes reduce or
eliminate the occurrence of undesired or adverse systemic
effects which are otherwise observed when the liposomes
are formed from the same lipid constituents but omitting
the electrically charged component are administered.
In particular, the addition of a component having a
negative charge, e.g., a fatty acid such as stearic acid,
SIJE~TI~U~ SHEET ~RULE 26)
~ WOgS112386 7 4 ~ ~ 6 P~ /0~66~
to a liposome preparation improves the host's tolerance
to the preparation significantly in comparison to when
the same preparation is administered without the nega-
tively charged component. The improved tolerance may be
reflected in the reduction or elimination of adverse
systemic effects produced by a liposome preparation in
which the electric charge component is absent. These
systemic effects include, e.g., hemodynamic effects, such
as hemodynamic depression, changes in blood pressure,
heart rate, systolic pressure, diastolic pressure, or ECG
intervals (PR, QRS, QT, QTc), reflex tachycardia, prema-
ture ventricular arrhythmias, increased respiration,
sedation, and also changes in the blood cell and platelet
counts, and death. It is recognized that this basic
principle of improving tolerance to an otherwise
deleterious structure by electrical-charge modification
may be applicable to reagents other than liposomes.
The present invention relates to an amount of
electrical charge which is effective in, e.g., reducing
hemodynamic effects, improving tolerance, or reducing
adverse effects, of a liposome preparation. The
ef f ective amount of electrical charge may be added to the
liposome preparation by forming liposomes in the presence
of the effective amount of an electrically charged
component or by adding the effective amount of charge to
already formed liposomes.
The undesired or adverse effects of a liposome
preparation upon administration may be reduced or
eliminated by the addition of an electrical charge,
regardless of the composition of the a~;nictered
liposomes. For example, a liposome preparation having
systemic effects may already contain electrically-charged
components. In this case, the introduction of an
effective amount of an electrically-charged component may
be in addition to the charged components which are
already present in the liposome. The charged component
SlJBS117U~ SHEET ~RVLE 26~
WO9SI12386 2 ~ 74326 P~ O~C~ ~
introduced may be of the same type or charge already
present in the liposome or of an altogether different
type or charge. Thus, the present invention relates to
the addition of a charge to a liposome already having
charged components.
The charged component may be incorporated into the
liposome in various ways. By incorporating an
electrically charged component into a liposome, the
charged component may become, e.g., a structural
component of the lipid bilayer, and/or conjugated or non-
covalently attached to a constituent of the liposome.
For example, when liposomes are formed from a mixture
comprising an effective amount of a negatively-charged
stearic acid, the stearic acid may be incorporated into
the lipid bilayer of the liposome. However, the
effective amount of an electrically-charged component may
also be added to an already formed liposome preparation,
or an incompletely formed preparation, e.g., by
conjugating or non-covalently attaching a charged
component to the surface of the lipid bilayer.
How the addition of the electrically-charged
component to the liposome is accomplished is of minor
interest. The decisive feature is that the electrically-
charged component is present in the liposome in an amount
which is effective, e.g., to improve host tolerance,
reduce adverse effects, and/or reduce the hemodynamic
effects produced by the administration of a liposome
preparation. The electrical-charge can therefore be
carried and incorporated into the liposome in a variety
of ways, including, e.g., by the addition of charged
components such as acids, e.g., stearic acid, and salts
of these components such as ammonium salts; by coupling
charged moieties to components of the lipid bilayer; and
by the addition of bilayer-forming substances with
charges, either coupled covalently or adhering by any
other force. While the invention is not bound by any
S(JBST~7UrE SHEET ~RULE 26)
21 7~326
WO9S/12386 P~~ 1/03668
-- 7
theory, typically a liposome according to the present
invention presents an electrical-charge on the outer
surface of its bilayer.
For example, a liposome preparation comprising
S phosphatidyl choline is known to have an adverse
hemodynamic effect. Its tolerance by the host organism
may be improved by preparing a new batch of liposomes
comprising an effective amount of an electrically-charged
component in addition to the phosphatidyl choline, e.g.,
the effective amount of electrical charge may be carried
by stearic acid. Alternatively, the charge may be
introduced into liposomes which are already formed. For
example, an electrical-charge may be introduced to the
outer (exterior) surface of the lipid bilayer by chemical
modification, or by covalently or noncovalently adding a
charged moiety to its surface.
The addition of a charge to a liposome preparation
may also permit higher amounts of the preparation to be
administered to the host organism without producing dele-
terious effects normally attributed to liposome admini-
stration. This is significant in that larger dosages of
the agent encapsulated in the liposome may be attained.
The addition of a charge to a liposome may also improve
the stability and storage of the liposome preparation,
e.g., at temperatures greater than room temperature, such
as 30 or 40C.
The nature of the effective amount of electrically
charge component may be either positive or negative, as
long as the desired effect is achieved, e.g., to improve
tolerance, reduce adverse effects, and/or reduce the
hemodynamic effects produced by administration of a
liposome preparation. A negative charge is preferred.
The charge may be, e.g., ionic or electronegative. In
principle, once it is known that a particular liposome
preparation is not tolerated by the host animal, e.g., as
manifested by undesirable systemic affects, a charged
St~STlTUt~ SHEF~ rRUL~ 2fi~
WO95/12386 2 1 74326 P~ g~/03668 ~
component may be selected and then incorporated into the
liposome preparation for the purpose of reducing or
eliminating the side effects associated with its
administration. By preparing a series of liposome
preparations having various amounts and kinds of charge,
it would be routine to administer and determine how much
and what kind of charge is effective in, e.g., reducing
hemodynamic effects and improving host tolerance.
The amount of effective electrical charge may be
carried by a molecule or a charge carrier. The molecule
may have one or more charged moieties, e.g., a
zwitterion. The charge may be located at any position on
the charge carrier. The choice of the charge carrier
will depend on various factors; e.g., availability,
biocompatability, and desired effect. The charge carrier
may be selected for other properties in addition to its
ability to carry the desired charge. For example, the
charge carrier may also function as a targeting agent,
i.e., directing the liposome selectively to a desired
tissue or cell type in the host organism. The charge
carrier may be, e.g., biomolecule, a polymer, a lipid, a
protein, a nucleic acid, a carbohydrate, a synthetic
molecule, or any molecule or molecular structure which
may be incorporated into the liposome bilayer and which
possesses an effective amount of electrical charge. By
incorporated the molecule into the liposome bilayer, the
molecule may become structurally part of the liposome
vesicle, e.g., integrated into the lipid bilayer, or
peripherally attached to the liposome surface, or as a
lipid constituent, itself. Additionally, small compounds
with an additional acidic group can be coupled to an
"anchor" such as cholesterol, phosphatidyl choline, fatty
acid etc. sitting in the bilayer. The charge, preferably
negative, can also be carried by amino acids or sugar
acids coupled to any moiety able to adhere to the bilayer
(cholesterol, phosphatidyl choline, fatty acid, etc.).
SIJB~I~U~E SHEET 7RULE 26~
W09S/12386 ~l 74326 ,~"~q~,03668
. g
The charge carrier may also be any lipid which
carries a charge, e.g., including, sterols, fatty acids,
- glycerol esters, sphingosine, terpenes, or generally
lipids, e.g., see i~ ~OOK OF CLINICAL C~E~ISTRY (1986), N.W.
5 Tietz, editor, W.B. Saunders Co., Chapter 7, pp. 829-900.
Stearic acid is a preferred example of a fatty acid
having a negative electrical charge which may be employed
in the present invention in an effective amount, e.g., to
reduce hemodynamic effects of a parenterally administered
liposome preparation. Other fatty acids may include,
e.g., palmitic acid, arachidonic acid, linolic acid,
linoleic acid, and/or mixtures thereof.
The charge carrier may also be a protein which,
e.g., is capable of integrating into the lipid bilayer or
attaching peripherally to the surface. The charge of the
protein may be produced by amino acids, carbohydrate
linkages, or non-protein groups linked or joined to the
protein. The protein may also be a protein having a
lipid moiety. A protein may be chosen because of its
desired charge and also because of its ability to target
liposomes to a specific tissue.
The amount of charge to be incorporated into the
liposome may be that amount which is effective to reduce
the undesired systemic effects such as hemodynamic
depression and/or tolerance. The amount of charge may be
varied by changing the molar ratio between the charged
component and the other constituents of the liposome.
For example, a charged component may be added to a
liposome comprising phosphatidyl choline by the addition
of stearic acid. The amount of charge in the liposome
may be achieved by adjusting the molar ratio between the
two constituents to a desired amount; e.g., phosphatidyl
choline/stearic acid, 9:1.
The improved tolerance by the host to a liposome
preparation and the consequent elimination or reduction
in systemic affects may be produced by the incorporation
SlJBSTITl~E SHEET rRl)LE 26)
2 1 7~3~6
WOgS/12386 ~ 9]/03668 ~
-- 10 --
of a charged component into the liposome vesicle. This
improvement, when mediated by changes to the cell surface
caused by the charged component, may also be produced by
using agents or methods which modify surfaces. For
example, the already formed liposomes having a
deleterious systemic affect, may be treated enzymatically
or chemically to alter the surface properties of the
liposome and improve its tolerance when administered.
Chemical modification may include e.g., reacting the
liposome reducing agents, oxidizing agents, or coupling
charged components to its surface. These methods are
accomplished conventionally but to achieve the reduction
or elimination of adverse effects as revealed here.
The liposomes may be prepared according to methods
which are conventional in the art, e.g., as reviewed in
W086/00238. Additionally, liposomes may be prepared
according to EP69307; U.S. Pat. No. 5,110,475 which,
e.g., describes a process for the production of an
aqueous dispersion involving removal of liquid(s) from an
optionally multiphase liquid mixture by means of membrane
distillation; and W086/00238 which is described as an
extrusion techniques for producing liposomes having
substantially a unimodal and defined size distribution.
When a liposome is prepared with an electrically-
charged component, it is possible that the electrically-
charged moiety may be oriented in the liposome in several
different directions; e.g., facing the interior of the
liposome vesicle and facing the exterior at the interface
between aqueous medium and the lipid. Liposomes having
the electrical charge on the exterior are preferred,
e.g., the exterior surface. It may be desirable to
separate, e.g., the "interior" charged from the
"exterior" charged liposomes. This may be accomplished
by any mean as known in the art, including column
chromatography employing, e.g., charged or otherwise
modified supports, electrophoresis, electrical separation
SlJBSr~UrE SHEET fRULE 26)
~ WO9Stl2386 2 ~ 7 4 3 2 6 ~ 7C6~
-- 11 --
processes, and other processes which separate structures
based on charge, charge density, or charge orientation.
Generally, the liposome preparations containing the
electrically-charged component may be subjected to
S conditions which eliminate orientations which are not
desirable for the purposes of the present invention.
An active agent may be encapsulated in the liposomes
according to conventional methodology. ~hus, a liposome
preparation having an effective amount of an electrically
charged component may comprise or encapsulate an active
agent. Encapsulation may be accomplished by methods
which are known, e.g., by forming the liposome in the
presence of an amount of an active agent under conditions
in which the lipid bilayers form around and encapsulate
the active agent, by introducing an active agent to an
already formed liposome by fusion with a liposome, cell,
or other structure comprising the active agent, e.g.,
such fusion may be carried out by PEG, electric current,
lectins, and other well known methods in the art. In
addition, liposomes having an active agent may be
prepared according to Wo/89593, e.g., ~y treating the
liposomes in an a~ueous medium under pressure conditions
effective to reduce the order of the lipid arrangement
therein to permit entry of the active agent and
performing a pressure drop.
An active agent may be encapsulated into a liposome
preparation having an effective amount of an electrically
charged component, including e.g., conventional active
agents such those used for therapy, including agents as
listed in the Physicians Desk References, Medical
Economics Data, 44th Edition, 1993 or Remington's
Pharmaceutical Sciences, Mack Publishing Co., 18th
Edition, 1990, antibodies, peptides, DNA, RNA, ribozymes,
and oligonucleotides, prophylaxis, and diagnostics, e.g.,
contrast agents, such as iopromide, paramagnetic
entities, ferromagnetic entities, radioisotopes, and
SlJBSrl7UTE S~EET IP~ULE 26)
2 ~ 7~326
WO9S112386 P~ 31/03668
- 12 -
other conventional structures or compounds. Such an
active agent may be one that is used and recognized by
the ordinary skilled worker in the field.
~The liposomes may be administered according to
conventional methods. For example, a liposome
preparation may be administered parenterally, e.g.,
intravenously by arterial or venous catheter, by
injection with a syringe, however they may also be
administered orally or through the intestines. A typical
amount of liposome preparation is, e.g., 100-200 mg
lipid/kg, this amount being dependent on how much of the
agent is intended for administration and how much of it
is encapsulated per liposome. The amount of liposome
preparation which is to be administered may be determined
routinely, according to methods which are known in the
art. The liposomes may be administered in a single
injection or it may be administered or infused over a
period of time at a constant or variable rate, e.g., 2
ml/min for 30 minutes. Because the liposomes comprising
the charged component are well tolerated by the host,
infusion rates and amounts of liposome to be administered
may be increased over the amounts that are typically used
for liposome preparations which have adverse effects.
The liposomes may be administered to any organism,
e.g., ~ ls, such as humans, rats, mice, dogs, cats,
rabbits, cows, horses, but also birds, ~phihians, and
reptiles. The purpose of administration may be to
diagnose, treat, medicate, sedate, or prevent disorders
but it may also be used in conjunction with the
development of animal models to evaluate the safety and
efficacy of drugs, contrast media, antibodies, and other
agents for their eventual use in humans. The method of
the present invention of e.g., reducing hemodynamic
effects, improving host tolerance, or reducing adverse
effects, associated with liposome administration, may be
accomplished in any organism, such as those listed above.
S~BSrlTlrrE SHEET ~RULE 261
W09S/12386 - 13 ~ S~ 6
The liposomes can be used for any diagnostic or
therapeutic use, including e.g., MRI, Ultrasound, and
~ Nuclear Medicine.
Without further elaboration, it is believed that one
skilled in the art can, using the preceding description,
utilize the present invention to its fullest extent. The
following preferred specific embodiments are, therefore,
to be construed as merely illustrative, and not limita-
tive of the remainder of the disclosure in any way
whatsoever.
In the foregoing and in the following examples, all
temperatures are set forth uncorrected in degrees celsius
and unless otherwise indicated, all parts and percentages
are by weight.
The entire disclosures of all applications, patents
and publications, cited above and below, are hereby
incorporated by reference.
SuBsrmn~ SHEET~RVLE~
WO9S/1~86 2 ~ 74 ~2~ /03668 ~
- 14 -
E X ~ M P L E S
ExamPle 1
The preparation process for iopromide liposomes by
the "ethanol vaporation method" is depicted in Figure 1.
An ethanolic solution of the membrane lipids (standard =
92.5 mg/ml phosphatidylcholine/cholesterol/stearic acid
4:5:1 molar) is mixe~d with an iopromide solution which is
obtained by diluting aliquots of an Ultravist~ 370 solu-
tion with 20 mM trome~h~;ne buffer, pH 7.4 to give a
final iodine concentration of 46.25 mg/ml. After the
mixing period, ethanol is removed from the mixture under
reduced pressure until a white liposomal suspension is
obtained. This suspension is then filtered (5 ~m and 1.2
~m), filled into vials and lyophilized to produce a
stable form for storage.
Prior to application, such lyophilizates are first
rehydrated with 4 ml 135 mM mannitol solution per g of
dry substance. The resulting liposomal suspension is
then filtered (20 ~m glass-fiber pre-filter and 5 ~m CN
main filter) to remove non-liposomal structures. These
structures are sling- or loop-shaped bilayer formations
in part larger than 10 ~m. It has been shown that these
structures are removed by the above filtration step.
The suitability of this preparation process for re-
producible production of larger amounts of liposomes hasalso been demonstrated. Preparation of 16 batches (1000
ml liposomal suspension each) resulted in encapsulation
of 42.1 + 4.9% and a mean diameter of 458 + 55 nm in the
resuspended material (2 ml 135 mM mannitol solution per g
lyophilizate).
The process is potentially suited to performance
under aseptic conditions. The solutions emplo~ed to
produce the liposomes can be ultra- or sterile-filtered.
The composition of a resuspended lyophilizate (4 ml
135 mM mannitol solution per g lyophilizate) is listed
below (without ranges):
SlJBS~lTU~ SHEET /RUL E 2
~ W09S112386 2 ~ 74326 P~ ,03668
- 15 -
iopromide: l~5.5 mg
phosphatidylcholine (PC): 41.3 mg
DL-~-tocopherole: 0.04 mg
cholesterol (CH):25.0 mg
stearic acid (SS):3.7 mg
tromethA~;ne: 3.7 mg
l N HCl: 25.4 mg
disodium edetate:0.15 mg
mannitol: 2l.8 mg
water f.i.: ad l ml
A molar lipid mixture of PC/CH/SS 4:5:l resulted in
the highest degree of encapsulztion (42.5 + 3.7%, n = 6)
as well as the smallest mean diameter (439 + 87 nm) in
the resuspended material. These values are in close
agreement with the values that were obtained when the
batch size was increased lO-fold (see 3.l). The molar
ratio of three membrane components as well as the type
and quality of each lipid component were optimized
regarding these target parameters.
The use of other non-ionic monomer contrast agents
(iopamidol, iohexol and ZK 119095) rather than iopromide
did not prove superior with regard to iodine encapsula-
tion. When the non-ionic dimer iotrolan was encapsu-
lated, higher weight ratios (mg iodine/mg lipid) were
achieved. This compound was excluded from further
studies, however, because of unacceptably long retention
of liposomal iotrolan in the liver and spleen of test
animals (biodistribution studies) and concomitant
pathological changes in the liver.
In the ethanol-evaporation process an iodine/lipid
ratio of l:l in the original solutions (see Fig. l)
proved the most suitable as regards optimal use of the
lipid (i.e., with the highest possible iodine to lipid
weight ratio) in the final preparation. Of the resuspen-
sion media tested, 135 mM mannitol resulted in the high-
est encapsulation values in comparison with bidistilled
SlJBSrlTUT~ SHEET ~RVLE 26~
WO9S112386 2 ~ ~ ~ 3 2 ~ ~ i i~l 3~ 6~ ~
- 16 -
water, various buffer solutions and mannitol solutions of
lower concentrations.
Example 2
For an initial stability study, a batch of liposome
lyophilizate was produced under aseptic laboratory
conditions. The resuspended liposomes were characterized
regarding size (QUELS), iodine encapsulation, pH and
microscopic appearance. The microbial status was not
determined. According to the results obtained in this
study (six months' storage), liposomal lyophilizates
(PC/CH/SS 4:5:l) can be stored at temperatures up to 30OC
without perceivable quality changes. After 6 months at
40C, however, a significant increase in size and a
decrease in pH were observed in the resuspended material.
These changes were accompanied by an unpleasant odor
coming from the resuspended material.
In a stability study of the (unfiltered) resuspended
material, no significant changes were observed in size
(QUELS), encapsulation or pH over a period of 24 h.
Possible changes in the microscopic appearance of the
preparation were not monitored in this study, however;
and so, the microscopic appearance of a resuspended
lyophilizate was monitored separately both prior to and
after filtration over a time-period of 8 hours. No
significant changes were observed in either of the
materials ~XAm; ned.
Example 3
~A) Methods
(i) Sur~ical Procedure~
Six to seven days prior to study, 6 normal healthy
adult mongrel dogs of either sex were selected for
similarity in weight (14.3-15.6 kg) and anesthetized with
35 mg/kg, iv pentobarbital sodium, shaved, and prepared
SlJBSrlTl~ SHEET IRULE 26)
~ WO9Sl12386 2 1 71~3 2 6 ~ 03668
for sterile surgery by washing the area of the intended
incision twice with Betadine~ surgical scrub, and then
painting the area with Betadine~ solution. The dogs were
intubated using a cuffed endotracheal tube and
mechanically respired with room air using a Harvard
animal respirator (model 607), placed in dorsal
recumbency on the surgical table and draped for surgery
using sterile techniques. A small (-l inch) incision was
made between the manubrium of the sternum and the greater
tubercle of the right humerus. Using blunt dissection,
the right omocervical artery and right jugular vein were
isolated for cannulation. The artery was cannulated
first using Tygon microbore tubing (0.05" ID, #S-54-HL).
The catheter was secured with 2-0 silk suture and
exteriorized via a subdermal tunnel to the nape of the
neck. In a similar fashion, the right jugular vein was
catheterized using Tygon microbore tubing (0.04" ID, #S-
54-HL) and exteriorized to the nape of the neck also.
Both arterial and venous catheters were filled with
sterile, heparinized (600 U/mL) saline, sutured and
painted with betadine to reduce infection. The exposed
catheters were secured in a small plastic pouch and
enclosed in a cotton-mesh collar reinforced with adhesive
tape. Dogs were administered standard intramuscular
injections of Com~iotic to reduce the risk of infection
and allowed to recover from surgery.
~i) Ex~erimental Procedures
On the day of study, the dogs were brought into the
laboratory two at a time in individual mobile slings.
The dogs were shaved at the ankles and instrumented with
lead II electrocardiographic (ECG) leads using surface
electrodes and conducting jelly. The specialized collars
were removed, blood was withdrawn from each catheter and
the catheters were flushed with sterile saline. The
arterial catheter was attached to a pressure transducer
SuBsTmn~ SHEET~RULE2~
WO9S112386 2 ~ 74~26 PCT~4/03668 ~
- 18 -
and cali~rated using a mercury manometer. A lO mL sample
of venous blood was collected from the venous catheter
using sterile Sarstedt monovette tubes containing EDTA.
Following an equilibration period of 20-40 minutes, dogs
were administered an infusion of DMPC/DPPC, prepared by
the interdigitation/fusion method (See Wo 9l/10422; EP
510096), or DPPC at 2 mL/minute for -25 minutes.
Parameters monitored were direct arterial blood pressure
and lead II surface ECG.
~iii) Druq Administration
DMPC/DPPC or DPPC liposome vesicles were admini-
stered intravenously as an opa~ue suspension at a rate of
2 mL/minute in a concentration of l mg/kg/mL until a
total dose of 50 mg/kg was administered. All doses were
lS loaded into sterile 60 mL syringes and infused using a
Razel infusion pump. Any remaining suspension in the
catheter was flushed into the dog using sterile saline at
the same infusion rate.
(ivl Statistical AnalYsi~
Selected CBC counts after vesicle administration
were compared to basal values using a paired sample
t-test. Differences were considered significant when
P S 0.05.
~B) Results
The effects of DMPC/DPPC or DPPC infusions on heart
rate (HR), mean blood pressure (MAP), systolic pressure
(SP), diastolic pressure (DP), ECG intervals (PR, QRS,
QT, QTc), and selected blood cell count values are shown
in tables 1-4. The complete blood cell count (CBC)
values are contained in the appendix.
SlJBSTl~UrE SHEET ~RULE 26~
~= ~ -
2 1 74~6
WO95/12386 ~ /03668
- 19 -
~i) Blood Pressure
Intravenous administration of DMPC/DPPC or DPPC
vesicles caused a marked decrease in systolic, diastolic,
and mean arterial pressure.
~ii) SYstolic Pre~sure (Table l)
Systolic pressure fell an average of 37% during
infusion of DMPC/DPPC. An even greater fall in systolic
pressure was observed during the infusion of DPPC
vesicles with systolic pressure dropping 55%. While
systolic pressures returned to near baseline levels in
only one dog from each group during observation period,
this pressure fall was determined to be transient as
systolic pressures were within normal physiological
levels within 30 minutes following vesicle
administration.
(iii) ~iastolic Pressure ~Table 2)
Diastolic pressure fell 39% in the DMPC/DPPC group
and 48% in the DPPC group during the vesicle infusion.
One dog from the DMPC/DPPC group maintained control
levels of diastolic pressure during the infusion and l
dog from the DPPC group demonstrated m in;~l effects on
diastolic pressure. On the average, the effect of
liposome vesicles on diastolic pressure was transient, as
mean diastolic pressures returned to near control levels
within 30 minutes following vesicle administration.
~iv) Mean Pressure (Table 3)
The effect of liposome vesicles on mean arterial
pressure was clearly more marked in the DPPC ~roup with
lower and more prolonged falls in pressure. The dogs in
the DMPC/DPPC group experienced an average fall in mean
pressure of 38% during infusion, but returned to near
control levels by the end of the infusion. Dogs re-
ceiving DPPC vesicles, however, recorded a 52~ fall in
SlJB~TUTE StlEET ~RVLE ~6~
WO95112386 2 1 7 4 ~26 ~ 9~ Y ~
- 20 -
mean pressure during infusion and did not recover to near
control levels until 30 minutes following the end of the
infusion.
~v) Heart Rate (Table 4)
The effect of liposome vesicles on heart rate in
conscious dogs was variable. While mean heart rate
values increased in both groups during administration of
liposome vesicles, this reflects a pronounced tachycardia
recorded from only one dog in each group. The other 2
dogs in each group maintained a heart rate equal to or
lower than control during the infusion and the 45 minute
observation period that followed.
~vi) Electrocardio~raPhic Intervals lTable 5)
The infusion of DMPC/DPPC or DPPC liposome vesicles
had no physiologically marked effect on ECG intervals.
No changes were observed in PR or QRS intervals (Table 3A
and 3B, pg. 13). Minor increases in QT (Table 3C, pg.
14) and QTc (Table 3D, pg. 14) were recorded but did not
reflect a major physiologic effect sufficient to be
significant.
~vii) CBC Values ~Table 6)
The infusion of DMPC/DPPC liposome vesicles
increased white cell count significantly and lowered
circulating platelets slightly, red cell count and
hematocrit were not effected (Table 4A, pg. l5). The
infusion of DPPC vesicles also had little effect on red
cell count and hematocrit, however did significantly
lower platelet count while lowering white cell count
slightly (Table 4B, pg. 15).
~C) Di~cu~ion
The present study, in which conscious dogs were
monitored for acute hemodynamic and ECG changes, showed
~lJBSrllU!rE SHEET ~RUL~ 26)
~ W09S/12386 2 ~ 7 ~ 3 ~ 6 pCT~4/03668
- 21 -
the intravenous infusion of DMPC/DPPC and DPPC liposome
vesicles caused a sudden and significant drop in blood
pressure resulting in a variety of physiologic reactions
including lethargy, and mild sedation in all dogs with
S emesis occurring in only one dog. No physiologically
significant changes were noted in ECG parameters;
however, white blood cell counts and circulating platelet
counts were effected.
The majority of these overt reactions could be
attributed to the transient, but significant, decrease in
blood pressure resulting from a possible anaphylactic
reaction to the test substances. Accompanying such a
reaction would be changes in white blood cell and
circulating platelet counts.
Example 4
IA) Methods
(i) Surqical Procedures
Six to seven days prior to study, 5 normal healthy
adult mongrel dogs of either sex were selected for
similarity in weight (13.7-14.l kg) and anesthetized with
35 mglkg, iv pentobarbital sodium, shaved, and prepared
for sterile surgery by washing the area of the intended
incision twice with Betadine~ surgical scrub, then
painting the area with Betadine~ solution. The dogs were
intubated using a cuffed endotracheal tube and
m~chAnically respired with room air using a Harvard
animal respirator (model 607), placed in dorsal
recumbency on the surgical table and draped for surgery
using sterile techniques. A small (l inch) incision was
made between the manubrium of the sternum and the greater
tubercle of the right humerus. Using blunt dissection,
the right omocervical artery and right jugular vein were
isolated for cannulation. The artery was cannulated
first using Tygon microbore tubing (0.05" ID, #S-54-HL).
The catheter was secured with 2-0 silk suture and
SlgBSTl~UTE Sl IEET ~RUL~ 26~
2 1 7~26
WO9S11~86 P~ /03668
- 22 -
exteriorized via a subdermal tunnel to the nape of the
neck. In a similar fashion, the right jugular vein was
catheterized using Tygon microbore tubing (0.04 inch ID,
#S-54-HL) and also exteriorized to the nape of the neck.
Both arterial and venous catheters were filled with
sterile, heparinized (600 U/mL) saline (l mL) and capped
with sterile caps. The 2 incisions were closed with silk
suture and painted with Betadine~ to reduce the risk of
infection. The exposed catheters were secured in a small
plastic pouch and enclosed in a cotton-mesh collar
reinforced with adhesive tape. Dogs were administered
st~n~Ard intramuscular injections of Combiotic to further
reduce the risk of infection and allowed to recover from
surgery.
(ii) ExPerimental Procedures
On the day of study, the dogs were brought into the
laboratory in individual mobile slings. The dogs were
shaved at the ankles and instrumented with lead II
electrocardiographic (ECG) leads using surface electrodes
and conducting jelly. The specialized collars were re-
moved, blood was withdrawn from each catheter and the
catheters were flushed with sterile saline. The arterial
catheter was attached to a pressure transducer and
calibrated (0-200 mmHg) using a mercury manometer. A l0
mL sample of venous blood was collected from the venous
catheter using sterile Sarstedt monovette tubes contain-
ing EDTA. Following an equilibration period of 20-40
minutes, dogs were administered an infusion of EPC at 2
mL/minute for 25 minutes. Parameters monitored were
direct arterial blood pressure and lead II surface ECG.
~iii) Drug Administration
EPC liposome vesicles were administered intraven-
ously as an opaque suspension at a rate of 2 mL/minute in
a concentration of l mg/kg/mL until a total dose of 50
SHEET IRULE 26~
WosS/l~86 ~,1 7~3~ i~,S/03668
- 23 -
mg/kg was administered. All doses were loaded into
sterile 60 mL syringes and infused using a Razel infusion
- pump. Any remaining suspension in the catheter was
flushed into the dog using sterile saline at the same
infusion rate to assure that the total dose was
a~in;ctered to each dog.
tB) Result~
The effects of EPC liposome vesicle infusions on
heart rate (HR), mean blood pressure (MAP), systolic
pressure (SP), diastolic pressure (DP), ECG intervals
(PR, QRS, QT, QTc), and complete blood count values (CBC)
are described below. Peak changes in blood pressure and
heart rate occurred during the infusion of liposome
vesicles.
~i) Blood Pressure
Intravenous administration of EPC liposome vesicles
caused a marked decrease in systolic, diastolic, and mean
arterial pressure in 3 of the 5 dogs tested.
tii) SYstolic Pre~sure (Table 7)
Systolic pressure fell an average of 36% in a group
of 5 dogs. Those dogs experiencing acute hemodynamic
depression (n ~ 3) had systolic pressure dogs ranging
from 50% to 62% below basal levels. These changes in
systolic pressure were transient as systolic pressure
values returned to normal levels by the end of the EPC
infusion.
(iii) Diastolic Pressure ~Table 8)
Diastolic pressure fell 36% among the 5 dogs tested.
Those 3 dogs which experienced a marked decrease in blood
pressure had diastolic pressure drops ranging from 56% to
61% below basal levels. While these drops in pressure
were severe, they were also transient as pressure values
SlJBSrl7UTE SHEET ~RVLE 2~
WO9SI1~86 ~ 2 6 P~ 9~/03668
- 24 -
returned to near basal levels by the end of the infusion
of the EPC vesicles.
~iv) ~ean Pres~ure tTable g)
The infusion of EPC liposome vesicles had no ob-
served effect in 2 of the 5 dogs tested; however, 3 ofthe S dogs tested demonstrated marked falls in mean
arterial pressure. The average fall in mean pressure was
35~, but those dogs with marked hemodynamic depression
experienced mean pressure drops ranging from 57% to 61%
below basal levels. While these drops in pressure were
severe, they were also transient as pressure values
returned to near basal levels by the end of the infusion
of the EPC vesicles.
(v) Heart Rate ITable lO)
The effect of EPC liposome vesicles on heart rate in
conscious dogs was closely related to physiologic re-
sponse to falls in blood pressure. Those dogs which were
compromised hemodynamically by the EPC infusion demon-
strated a profound reflex tachycardia during EPC infusion
followed by brief bradycardia, with heart rate returning
to basal levels in all dogs by 30 minutes post EPC infu-
sion. The 2 dogs which did not experience hemodynamic
compromise during EPC infusion maintained relatively
constant heart rates throughout the study.
(vi) ElectrocardioqraPhic Intervals (Table ~, A-D)
The infusion of EPC liposome vesicles had no physio-
logically significant effect on ECG intervals. No
changes were observed in QRS duration. Changes occurring
in PR, QT and QTc intervals were related to heart rate
changes that occurred in the 3 dogs overtly affected by
the EPC infusion. None of these changes were determined
to be of physiologic significance due to their transient
nature.
g~lTU~ SHEET~RV~E2
== ~ ~ ~
~ WO9S/12386 2 J 7 ~ ~ ~ 6 ~ /03668
- 25 -
In one dog, spontaneous arrhythmias (in the form of
isolated premature ventricular contractions) occurred
during the EPC infusion. Examples are shown in Figure 2.
(vii) Mean CBC Value~ (~able 12)
The infusion of EPC liposome vesicles did not effect
red blood cell count or hematocrit. White blood cell
counts were not consistent. Platelet counts were clearly
depleted in those 3 dogs which experienced hemodynamic
depression. One of the dogs not experiencing hemodynamic
depression (USDA #97684) did experience a platelet
depletion, but not below normal range values.
(C) Discussion
Intravenous administration of EPC liposome vesicles
in conscious dogs resulted in hemodynamic depression.
The platelet depletion observed in this study paralleled
a similar effect noted in the previous study. In both
studies, platelet depletion seemed closely related to
severe falls in blood pressure resulting from liposome
infusion. The overt reactions observed during liposome
infusion (platelet depletion, marked hypotension, and
reflex tachycardia) are most likely related to an
anaphylactic response. The spontaneous arrhythmias
observed in one dog receiving EPC vesicles may be a
response to the sudden hypotensive effect with a reflex
activation of catecholamines.
Example 5
Liposomes according to the present invention,
comprising the electrically-charged component stearic
acid (preparations SA 504/02079 and SA 504/300591), were
administered to healthy dogs.
SLJBSrITU~E SHEET ~RULE 26)
WO9S/12386 2 1 7~26 - P~ 3668
- 26 -
(A) Methods
~i) Surqical Procedure~
Six to seven days prior to study, normal healthy
adult mongrel dogs of either sex were selected for simi-
larity in weight (12.3-15.1 kg) and anesthetized with 35
mg/kg, iv pentobarbital sodium, shaved, and prepared for
sterile surgery by washing the area of the intended inci-
sion twice with Betadine~ surgical scrub, then painting
the area with endotracheal tube and mechanically respired
with room air using a Harvard animal respirator (model
607), placed in dorsal recumbency on the surgical table
and draped for surgery using sterile techniques. A small
(1 inch) incision was made between the manubrium of the
sternum and the greater tubercle of the right humerus.
Using blunt dissection, the right omocervical artery and
right jugular vein were isolated for cannulation. The
artery was cannulated first using Tygon microbore tubing
(0.05" ID, #S-54-HL). The catheter was secured with 2-0
silk suture and exteriorized via a subdermal tunnel to
the nape of the neck. In a similar fashion, the right
jugular vein was catheterized using Tygon microbore
tubing (0.05" ID, #S-54-HL) and also exteriorized to the
nape of the neck. Both arterial and venous catheters
were filled with sterile, heparinized (600 U/mL) saline
(1 mL) and capped with sterile painted with Betadine~ to
reduce infection. The exposed catheters were secured in
a small plastic pouch and enclosed in a cotton-mesh col-
lar reinforced with adhesive tape. Dogs were admini-
stered standard intramuscular injections of Combiotic to
reduce the risk of infection and allowed to recover from
surgery.
~ii) Experimental Procedures
On the day of study, the dogs were brought into the
laboratory and placed in individual mobile slings. The
dogs were shaved at the ankles and instrumented with lead
~JBSrITUrE SHEET ~RUL~ 26)
~ WO9Stl2386 2 i 7 ~ 3 ~ 6 ~ 31~03668
II electrocardiographic (ECG) leads using surface
electrodes and conducting jelly. The specialized collars
were removed, blood was withdrawn from each catheter and
the catheters were flushed with sterile saline. The
arterial catheter was attached to a pressure transducer
and calibrated using a mercury manometer. A 10 mL sample
of venous blood was collected from the venous catheter
using sterile Sarstedt monovette tubes containing EDTA.
Following an equilibration period of 20-30 minutes, dogs
were administered an infusion of either SA 504/020791 or
SA 504/300591 at approximately 2 mL/minute for exactly 30
minutes. Parameters monitored were arterial blood
pressure and lead II surface ECG. Heart rate was
determined from the ECG tracing.
(iii) Dru~ Administration
SA 504/020791 or SA 504/300591 iodine-containing
liposome vesicles were administered intravenously as an
opaque suspension at a rate of approximately 2 mL/minute
at a final volume of 4.2 mL/kg and 4.5 mL/kg, respec-
tively. The infusion rate was adjusted such that thetotal infusion time was 30 minutes. All doses were
loaded into sterile syringes and infused using a Razel
infusion pump. Any remaining suspension in the catheter
was flushed into the dog using sterile saline at the same
infusion rate.
The test substances were prepared according to the
following procedure: SA 504/020791: 17.6 mLs of mannitol
solution (135 mM) was added to each vial of the test sub-
stance and allowed to sit for at least 10 minutes. Vials
were then shaken vigorously and allowed to sit for ano-
ther 10 minutes. After repeating the shaking procedure,
the suspensions were checked visually for agglomerates
(if agglomerates were present, the above procedure of
shaking the vials was repeated). Suspensions were then
drawn into 60 mL syringes and filtered through a sterile
E SHEET rRULE 2fi)
21 7~26
wos5ll2386 ~ /03668
- - 28 -
filter apparatus supplied by Schering AG. Each animal
received 4.5 mL/kg.
SA 504/300591: The procedure is exactly as outlined
above except that 18.0 mLs mannitol solution (135 mM0
were used per vial and each animal received 4.2 mL/kg.
tiv) Statistical ~nalY~i~
Hemodynamic parameters, ECG intervals, and selected
complete blood counts were compared to pre-treatment
basal values using a paired sample t-test. Differences
were considered significant when p S 0.05.
(v) LiPo~ome PreParation
SA 504/020791 and SA 5041/300591 were prepared
according to the following instructions.
A solution 1 was prepared by dissolving 54.6 gms of
lipoid S100, 33.0 gms of cholesterol, and 4.9 gms of
stearic acid in 9.5 ml of 96.5% ethanol. 2000 ml of a
solution 2 was prepared by combining 250 ml of iopromide
solution with 9.5 ml of 20 mM Tris HCl, pH 7..5, and 9.5
ml of 96% ethanol. While stirring, solution 1 was mixed
with solution 2. The ethanol was removed under vacuum
and the remaining solution was freeze-dried. 5 gm of
freezed-dried material was resuspended in 8.8 ml of 135
mmol mannitol. The resuspended liposome preparation was
typically injected at 300 mg iodine/kg.
tB) Results
The effects of SA 504/020791 or SA 504/300591 infu-
sions on heart rate (HR), mean blood pressure (MAP),
systolic pressure (SP), diastolic pressure (DP), ECG
intervals (PR, QRS, QT, QTc), and selected blood cell
count values are shown in Tables 13-16.
SlJBSrJ~U!rE SHEET IRULE ~6~
- : =
~WO9S/12386 2 ~ 7~3.~ 6 PCT~4/03668
- 29 -
(i) Blood Pre~sure ~Table 13)
Intravenous administration of SA 504/020791 or SA
- 504/300591 vesicles produced no significant changes in
systolic, diastolic or mean arterial pressures. As can
be seen in Table 13C, control mean arterial pressure for
dogs treated with SA 504/300591 was 104 + 6 and remained
relatively un~-h~nqed throughout the duration of the
experiment. Similar results were obtained with the test
substance SA 504/020791.
10~ii) Heart Rate
The liposome vesicle preparations had no significant
effect on heart rate in conscious dogs. Of the five dogs
treated with SA 504/300591, three dogs had a decrease in
heart rate during the infusion and two had an increase.
Thus, no consistent pattern was seen. In one animal
receiving SA 504/020791 (Dog #10003), the control heart
was particularly high (164 bpm). This animal was highly
reactive to any stimuli in the room (i.e., laboratory
personnel), but eventually seemed to quiet down as the
experiment progressed. This was most likely due to
acclimatization and not a drug-induced effect.
~iii) ElectrocardioqraPhic Intervals
The infusion of either test agent had no physiolo-
gically marked effect on ECG intervals. No changes were
observed in PR, QRS, QT, or QTC intervals.
(~ii) CBC Values
The infusion of either test agent had no significant
effect on RBC or WBC counts. Although the WBC values
were not significantly affected, the trend appears that
WBC will be increased following administration of the
test agents. If one pools the data from both groups
(i.e., n = 8), then statistical significance is achieved
for elevating WBC (p = 0.026). In 5 experiments,
SlJBS~UrE Sl~EET ~RULE 26~
2t ~32~
WO9511~86 P~ /03668
- 30 -
platelet values before and after the test substances were
categorized as either "adequate," "increased," or
"decreased." In all 5, the platelet values were
characterized as "ade~uate." In the remaining 3
experiments, values for platelet counts (thousands/mL)
are as follows:
Trea~nt Dog # C~ntrol 45'
SA 504/300591 10174 435 376
SA 504/020791 10003 198 243
SA 5Q4/020791 98701 210 298
___ _ ______ ____ _ ___ ___ ___ __ _ _ _ _
~C) Discussion
Previous studies on liposome preparations conducted
in conscious dogs with DMPC/DPPC, DPPC, and EPC liposome
vesicles demonstrated a dramatic, transient hypotensive
response in -40-60% of the animals tested. The present
study, in which conscious dogs were monitored for acute
hemodynamic and ECG changes, showed the intravenous
infusion of SA 504/300591 and SA 504/020791 liposome
preparations produced no significant changes in
hemodynamic variables (blood pressure and heart rate),
ECG intervals, or RBC and WBC values, and are therefore
well tolerated and free of adverse hemodynamic effects.
ExamPle 6
Two liposome formulations were prepared and tested
in a dog model for hemodynamic effects.
Preparation ': Liposomes which only contained phos-
phatidyl choline at a concentration of ca. 25 mg/ml.
~BSrITUrE SHEET rRVLE 26)
WO9S/12386 2 3 ~f~ 6 ~ SSI03668
- 3i -
Preparation 2: Liposomes with phosphatidyl choline
and stearic acid. The molar content was ca. 9:l at a
total lipid concentration of ca. 70 mg/hl.
These preparations were infused intravenously into
five dogs each at a dose level of ca. 50 mg lipid per kg
body weight (preparation l) and ca. 30 mg lipid per kg
(preparation 2).
The following effects were observed in the dogs.
Preparation ': Transient fall in blood pressure
associated with a significant reflex tachycardia in 3 of
the 5 animals tested. Other observed effects in these 3
animals included: premature ventricular arrhythmias dur-
ing the infusion and briefly following the administration
in l dog and increased respiration in the form of panting
in the 2 other dogs. There were no physiologically sig-
nificant changes in the ECG intervals in any of the dogs.
Platelet counts fell in 4 of 5 dogs receiving the prepa-
ration. Those dogs experiencing hemodynamic depression
seemed somewhat sedated and had the most consistent
platelet depletion when compared with those dogs who did
not experience decreases in blood pressure.
Preparation 2: Intravenous administration of this
formulation had no adverse hemodynamic effects in any of
the five animals treated.
Exam~le 7
In this example, the cardio-haemodynamic effects of
liposomes prepared from a saturated uncharged phospho-
lipid DSPC alone or in a combination with a negatively
charged phosolipid DSPG (9:l) were examined.
Preparation of liposomes: The liposomes which were
used in this study were prepared by a continuous high
pressure extrusion method.
g~mn~ SHEET~R~E2
2 1 7~26
WO9S11~86 PCT/~1~1/0~6~Y
- 32 -
Briefly, an ethanolic solution of the respective
lipid or lipid mixtures was deposited on the wall of a
round bottom flask. The resulting lipid film was
dispersed in 300 mM mannitol solution to give a lipid
concentration of approximately 100 mg/ml. The resulting
MLV dispersions were subsequently extruded using a high
pressure extrusion apparatus (Maximator~ model HPE 10.0 -
250, Schmidt, Kranz & Co., Zorge, Germany). Each batch
was sequentially extruded 10 times over two stacked
polycarbonate membranes (Nucleopore, TUbingen, Germany)
of decreasing pore sizes (1.0 - 0.4 and 0.1 ~m) to give a
total of 30 filter passages for each preparation. At the
end of the extrusion process the obtained liposomal
suspensions were filtered through sterile filter holders
(0.4 or 0.2 ~m pore size cellulose acetate, Sartorius,
G~ttingen, Germany) into sterile glass vials which were
stoppered under aseptic conditions.
~ipid Subst~nr6Q
All phospholipids were stored below - 20C and used
without further purification.
Distearoylphosphatidylcholine (DSPC- batches LP-04-013-
114219 and -116298) and Distearoylphosphatidyl-glycerol
(DSPG - batch LP 04-017-115751) were obtained from
Sygena, Liestal, Switzerland.
Liposome size was determined by photon correlation
spectroscopy (PCS) using a submicron particle-sizer,
autodiluterM model 370, Nicomp Instr. Corp., Santa
Barbara, CA, USA.
Osmolality [mOsm/kg] of samples and pH values were
determined with an automatic freezing point osmometer
(Knauer, Berlin, Germany) and a pH meter 761 Calimatic
(Knick, Berlin, Germany), respectively.
S(JBSTITUT~ SHEET ~RULE 26~
~ WO9S112386 2 ~ 7 4 3 ~ 3~103668
Animal experiments
Eighteen male rats (strain: Han-Wistar, breeder:
Schering SPF, standard feeding and housing conditions),
with a body mass of 360 to 430 g were randomly sorted
into three groups with n=6 animals per group.
The rats were anaesthetized with pentobarbital,
60 mg/kg intraperitoneally. The trachea was cannulated
to facilitate spontaneous respiration. Body temperature
was maintained at 38 + 0.5 C by means of a heated
operating table and a heating lamp.
Left ventricular pressure and blood pressure in the
femoral artery were recorded via polyethylene filled
catheters connected to Statham pressure transducers.
Cardiac output was determined by the thermodilution
method (thermistor catheter in the abdominal aorta,
injection of ice cold saline into the right atrium). The
animals were heparinized with an i.v. bolus of 400 U/kg.
The rats were set up to allow recording/calculation
of the following parameters: BPsyst. (systolic blood
pressure, mmHg), BPmean (mean blood pressure, mmHg),
BPdiast. (diastolic blood pressure, mmHg), HR (heart
rate, 1/min), C0 (cardiac output/100 g body mass,
ml/min/100 g), LVEDP (left ventricular enddiastolic
pressure, mmHg), TPR (total peripheral resistance,
dyne*s*cm-5103), ECG (electrocardiogram, ms) and dP/dt max
(maximum rate of left ventricular pressure rise, mmHg/s).
Surgery and instrumentation were followed by a 30-
min e~uilibration period, at the end of which the pre-
treatment values were determined. Data were recorded for
45 min after administration of the liposomes or mannitol
(volume control). Each animal received one treatment
only.
Rats were infused with a liposome dose containing a
total amount of 300 mg lipid/kg body weight and at an
injection rate of 100 mg lipid/kg/min into the left
femoral vein. Controls received an identical volume of
SlJ~SrlTUrE SHEE~ ~RVLE 26~
WO9S/1~86 2 1 7 ~ ~ 2 ~ sl03668 ~
- 34 -
300 mM mannitol solution. The investigated liposome
formulations were made from DSPC or DSPC/DSPG.
~tati~tics
Mean percentage changes of the haemodynamic data
versus prevalues were calculated (except for LVEDP, which
was expressed as absolute change from pre-drug baseline
values). Data were compared with the control group. The
Bartlett-test was carried out to test for equal variances
and one way analysis of variance (ANOVA) was used to
demonstrate statistical differences. Calculations of
statistical significance were performed using Student's
t-test. A p value below 0.05 was considered statis-
tically significant. Values in the figures are expressed
as mean percentage changes from prevalue + SEM.
RQsults
Control experiments
Mannitol 300 mM (volume control) did not cause
cardio-haemodynamic parameters to significantly differ
from prevalues. BPmean increased maximally by 1.8% (Fig.
2), BPsyst. by 2.3% and PBdiast. decreased by -2.2%
within the first 10 min post application. HR decreased
by -3.4%. TPR (Fig. 3) and LVEDP (Fig. 4) responded with
a short time increase by 5.5% and 2.8 mmHg, respectively
(Tab. 17). Contractility decreased by -3.3~.
DSPC liposomes
The use of the saturated DSPC resulted in neutral,
gel-state liposomes which led to marked cardiac and
haemodynamic disturbances when infused at a dose of 300
mg lipid/kg into rats. BPmean and TRP were significantly
decreased by -53.7% and -46.3%, respectively (Figs. 2 and
3). Contractility was reduced by -34.7% 5 min after the
SIJBSTI~U~ SHEET ~RUL~ 26~
~ WO95112386 2 i 7 ~ 3 2 6 ~ 1/03668
- 35 -
start of infusion. During this time LVEDP tFig. 4) was
significantly increased (p<0.05). All these effects were
accompanied by severe cardiac arrhythmia. There were
ventricular extrasystolies, ectopic beats, AV conduction
delays and failure of conduction to the ventricle.
However, HR and C0 were only slightly affected during the
first lO min post application. At the end of this lO min
period BP and contractility returned to baseline values.
HR, however, started to increase slightly above baseline
(about 10%) and contractility too. Until the end of the
observation period at 45 min p.a., contractility was
increased to +66.3% above baseline (p<O.Ol). TPR,
however, did not return to baseline by the end of the
observation time.
DSPC/DSPG lipo~omes
The addition of a negatively charged phospholipid
(lO mol%) to DSPC reduced the severity of the cardio-
haemodynamic side effects of DSPC significantly. BP and
TPR decreased after a short transient increase to -33.3%
and -35.5% of prevalue, respectively (Figs. 2 and 3).
Contractility declined by -14.3% during the first lO min
post application. It returned to normal values and was
increased at 30 min p.a. HR also responded with an
increase at later time points. Electrocardiographic
changes occurred in four of six rats during the liposome
infusion. The effects were interpreted as conducted and
nonconducted atrial premature beats in three animals and
in one animal as complex arrhythmia produced by paired
atrial premature beats. All ECG-effects disappeared
after the end of the infusion.
Thus, the addition of a negative charge to DSPC
liposomes significantly improved the cardio-hemodynamic
side effects of the preparation in rats.
The pr~ce~ing examples can be repeated with similar
success by substituting the generically or specifically
S~IT ~ SHEET~RVLE2~
W09~112386 2 ti ~3~6 P~ 3668 ~
described reactants and/or operating conditions of this
invention for those used in the preceding examples.
From the foregoing description, one skilled in the
art can easily ascertain the essential characteristics of
S this invention, and without departing from the spirit and
scope thereof, can make various changes and modifications
of the invention to adapt it to various usages and
conditions.
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W09S/12386 217~326 ~ 9q,03668
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A. PR (ms-c) VALUESIN EPC TREAI_3 D ~ S
U ~A~ . ~ASAL ~ 13'
97513 5 83 87 ~3 52
97682 93 135 128 105 38
97~92 ~7 lO' 87 85 P.
97804 119 ~15 116 120 119
97684 107 10 t 96 100 97
MEA~ - SEM 98-o 107=9 103=8 ~Ol~
B QRS (msec) VALUESIN EPC IREATED DO~S
~ ~.~ ~ ~A~A~ ~ : ~ ~ ~ .
97513 35 40 37 39 ~0
97682 30 30 33 33 33
97492 35 35 35 3_
9780. 33 33 3 3~ _~
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MEAN - SEM 33~1 34-~- 35 1 35_1 3~_1
SVBSTlnr~ SHEETfRVLE2~
W09S1123862 1 743~6 ~ s/03668 ~
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97492 1 ~3 228 188 170 180
97804 163 2~ 188 ' 70 180
97684 210 211 235 232 226
MEAN ~ SE M - 196=9 219=11217_11 20~ l l 202_7
OTC (msec) VALUES IN E?C TREAI'D DO~S
D.
97513 28g 293 276 299 28~
97682 270 233 298 320 283
97492 26c 260 227 2~1 224
9780. 267 296 261 289 302
9768. 2g2 2g8 275 287 273
MEAN _ SEM 277-5 276=13 267 12 285 15 273=13
SU~STmn~ SHEET ~RULE 2~
W0 95112386 2 1 7 ~ P~ ,03668
L r. D L~ - -
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7513 6 6 6
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MEAN ~ SE~ .4 1 43_1 ~l 5
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