Note: Descriptions are shown in the official language in which they were submitted.
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Schering AG 51229AWOMlXX00-P
Liposomes that Contain Contrast Media
for the Visualization of Intravascular Space
The invention relates to liposome formulations which contain
contrast media with a high intravascular retention period and
which are suitable for the visualization of intravascular space.
Prior Art
In recent years, liposomes have gained increasing importance
as potential vehicle systems for various types of contrast media.
The use of liposomal contrast medium formulations has been
described for all imaging diagnostic processes (diagnostic
radiology, computer tomography, MRI diagnosis, radiodiagnosis)
(Seltzer, St. E., Liposomes in Diagnostic Imaging, in:
Gregoriadis, G. (Editors), Liposomes as Drug Carriers, John Wiley
& Sons Ltd., Chichester, New York, Brisbane, Toronto, Singapore
1988, p. 509).
owing to their structure, the liposomes make it possible
both to incorporate hydrophilic contrast media into the aqueous
phase and to incorporate lipophilic contrast media into the
bilayer phase (Seltzer, St. E., Radiology 171, 19-21 (1989)).
After intravenous administration, liposomes preferably
accumulate in the organs of the mononuclear phagocytic system
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(referred to as MPS, and RES), whereby the highest concentrations
are reached in the liver and spleen. In the case of liposomes
that contain contrast media, this so-called passive targeting is
used to achieve selective concentration of these substances in
the healthy liver. Thus, it has been possible to achieve a
delineation of tumorous alterations of the liver in the rabbit
model with, for example, the liposomally encapsulated x-ray
contrast medium iopromide (Ultravist(R)) (Sachse, A. et al.,
Invest. Radiol. 28, 838-844 (1993)).
The in vivo behavior of liposomes can be influenced to a
large extent by altering the chemical composition as well as the
physical properties of the vesicles. The blood half-life and
organ distribution of the liposomes are influenced by parameters
such as vesicle size, surface charge (zeta potential), lipid
composition, and lipid dose (Senior, J. H., CRC Crit. Rev.
Therap. Drug Carrier Syst. 3, 123-193 (1987)). The blood half-
life of liposomes can thus be significantly prolonged by, for
example, reducing liposome size or making the membrane rigid by
using, e.g., saturated phospholipids (e.g., distearoyl
phosphatidylcholine, DSPC). The introduction of a charge can in
turn lead to a drastic increase in MPS uptake and thus to a
reduction in blood half-life. Moreover, the blood retention
period is very greatly influenced by the administered lipid dose
or number of particles. Higher lipid concentrations result in a
reduction of relative liver uptake, probably because of
saturation of the liver uptake mechanism. At the same time, a
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higher spleen uptake as well as a l~nger blood retention time are
observed.
With respect to therapeutic or diagnostic applications of
liposomes in the case of organs outside the MPS, there was great
interest in significantly prolonging the blood half-life of
liposomes. The desired purpose here was to be able to achieve,
i.a., enhanced extravasation of corresponding liposomes in areas
with damaged vascular endothelium (e.g., tumors or
inflammations). In recent years, it has been shown that the
blood retention period of liposomes can be altered by surface
modification of them (hydrophilization). In this case, mainly
lipid derivatives of polyethylene glycol (e.g., DSPE-PEGl900)
turn out to be advantageous compared to the polysaccharides or
glycol lipids (e.g., GM1) that were first used (Allen, T. M.,
Adv. Drug. Delivery Rev. 13, 285-309 (1994)). This effect of
PEGylation is attributed to the formation of a stearic barrier on
the liposome surface, which significantly alters the interaction
of the liposomes with various plasma components (e.g., plasma
protein or opsonin). When PEGs with molecular weights of between
1900 and 5000 are used, the blood half-lives of such sterically
stabilized liposomes (SSL) are in the range of about 9 to 16
hours. In this case, the liver uptake of corresponding liposomes
(100-200 nm) reached values of up to, for example, below
approximately 25% of the administered dose. It can be noted,
however, that the liver and spleen, as before, represent the main
organs for the uptake of SSL.
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If liposomal preparations are used for visualizing
intravascular space (blood pool imaging), it is necessary to
avoid the uptake of liposomes into the MPS as much as possible.
Owing to the relatively high blood volume, especially in the case
of CT, high contrast medium concentrations are required in the
vascular system to make reliable imaging possible. If, within
the framework of diagnostic studies in the area of the vascular
system, a concentration of liposomal contrast medium develops in,
for example, the liver and spleen, this can have an adverse
effect on the functions of the MPS (e.g., resistance to
immunity). In mice, significant impairment of the MPS uptake
capacity for carbon particles has been detected even with small
to average placebo-liposome doses (20-80 mg/kg) (Allen, T. M. et
al., Journ. Pharm. Exper. Therap. 229, 267-275 (1984)).
Moreover, the encapsulated contrast medium can also lead to
alterations of the RES.
As already mentioned above, liposomal contrast media have
already been incorporated into the development of organ-specific
contrast media. WO 88/09165 thus describes injectable, aqueous
liposome preparations with iodine-containing x-ray contrast
media, as well as a process for the production of corresponding
formulations. Owing to the size (0.15-3 ~m), as well as the high
contrast medium inclusions (iodine/lipid ratio of between 1.5 and
6 g/g), corresponding preparations should be especially suitable
for visualizing the liver.
In EP 0160552 A2, micellar or liposomal contrast media are
described for magnetic resonance tomography (MRT, MRI). The
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small unilamellar liposomes according to the invention (S W 60 +
10 nm) should lead to enhanced tumor concentration of the
liposomal Gd-DTPA after i.v. administration in tumor-bearing
mice .
In W0 90/04943, liposomal MRT contrast media, methods for
their production, and applications are described. The liposomes
according to the invention have an average diameter of less than
50 nm and should be suitable for visualizing the vascular system,
the heart, and the perfusion of tissues (blood pool imaging) in
addition to their application for visualizing tumors of the liver
and spleen. These small liposomes have the drawback, however,
that, owing to their limited volume, only small amounts of
hydrophilic components can be included. Also, depending on the
lipid composition of the liposome membrane in the case of MRI
contrast media, a significant reduction in the relaxivity of the
encapsulated components is described. More recently, therefore,
lipophilic paramagnetic chelates have gained increasing
importance. These components are incorporated into the liposome
membrane (bilayer) and therefore behave like membrane lipids with
regard to their pharmacokinetics. Corresponding liposomes
(memsomes) that have been additionally surface-modified
(PEGylated) to prolong their blood half-life should be especially
suitable for blood-pool imaging owing to their high relaxivity
(Tilcock, T., J. Liposome Res. 4, 909-936 (1994)).
The last-mentioned author also describes surface-modified
(PEG) liposomes for visualizing the vascular system in nuclear
diagnosis. In this connection, the radioactive component can be
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included either in the internal aqueous phase or the membrane
phase. Liposomes that are radiolabeled on the surface (PE-DTTA
and 99mTc), with an average diameter of about 100 nm, have a
blood half-life of more than 12 hours in the case of surface
hydrophilization with 4-6 mol% PE-PEG 6000 (SSL). After 8 hours,
high activity was obtained in the heart and the blood vessels
with appropriate preparations. At the same time, however, a
significant liver concentration was detectable.
The visualization of the vascular system has very great
importance in radiological practice. New contrast media for
specific visualization of vessels and the heart (e.g., particular
systems, macromolecules) should remain in the vascular system for
an extended period after intravenous injection. This "blood pool
effect" of new contrast media could make it possible to diagnose
more accurately with noninvasive methods many pathological
conditions, which, on the one hand, are characterized by
reduction in blood flow (e.g., by thrombosis, embolisms, tumors)
or, on the other hand, by an abnormal increase in blood flow
(e.g., by the disruption of capillary integrity). In addition,
accurate visualization of the perfusion of various tissues and
organs (e.g., heart, lung) or of pathological alterations in the
heart (e.g., valvular defects) would be achieved.
The attempts that have been made to date to produce
appropriate contrast media in CT and MRT have failed due to the
inadequate pharmaceutical or pharmacological properties of these
pharmaceutical carrier systems. Thus, it is necessary to be able
to produce in a reproducible manner appropriate contrast media
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with defined chemicophysical properties in large quantities.
Moreover, despite the need to administer large quantities of
contrast media (mainly for use in CT), very good compatibility of
the corresponding vehicle system has to be ensured to obtain a
positive risk/benefit assessment. Since the diagnostically
relevant study period for the visualization of intravascular
space is limited, for example, over the range of about two hours
after the injection (p.i.), appropriate contrast media should be
excreted as quickly and completely as possible. Also, there
should not be any excessive concentration in ranges that are not
diagnostically relevant with this indication.
For the visualization of intravascular space, there is
therefore a need for liposomal contrast media that avoid the
above-mentioned drawbacks, have a sufficient blood half-life, and
accumulate only to a small extent in the liver, spleen, or other
organs.
This object was achieved by this invention, especially by
the liposomal contrast medium formulations as they are
characterized in the claims.
The invention therefore relates to active ingredient-
containing liposome formulations, characterized in that
a) the following mixture ratio of lipids is present:
40-90% phospholipids or amphiphiles,
10-50% sterols,
0-25% charge carriers,
b) the liposomes have an average diameter of 100-400 nm
and
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c) the active ingredient is an x-ray or MRI contrast
medium or a radiodiagnostic agent.
The invention preferably relates to active ingredient-
containing liposome formulations, characterized in that
a) the following mixture ratio of the lipids is present:
40-70% phospholipids or amphiphiles,
30-50% sterols,
5-20% charge carriers,
b) the liposomes have an average diameter of 100-400 nm
and
c) the active ingredient is an x-ray or MRI contrast
medium or a radiodiagnostic agent.
The invention relates especially preferably to active
ingredient-containing liposome formulations, characterized in
that
a) the following mixture ratio of the lipids is present:
60-70% phosphatidylcholine,
20-30% cholesterol,
2-10~ phosphatidylglycerol, phosphatidic acid
and/or cholesterol hemisuccinate,
b) the liposomes have an average diameter of 150-350 nm
and
c) the active ingredient is an x-ray or MRI contrast
medium or a radiodiagnostic agent.
The processes or process steps that are suitable for the
production of contrast medium-containing liposome preparations
according to the invention can generally be considered to be
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among the standard methods that are-known in liposome technology
(e.g., New, R. R. C., Preparation of Liposomes, in: New, R. R.
C. (Editors), Liposomes: A Practical Approach, Oxford University
Press, New York, 1990). For the production of liposome
suspensions with the properties according to the invention,
however, continuous high-pressure extrusion is especially
suitable (WO 94/08626). Moreover, for example, the use of other
mechanical-dispersion or multiphase dispersion processes can
also, however, be carried out for the production of preparations
according to the invention. The liposome preparations according
to the invention that are produced in this way can be stored
directly or in freeze-dried form or kept on hand for use. The
latter samples are to be resuspended in each case before use.
In addition to the encapsulated portion of a hydrophilic
(water-soluble) contrast medium, the liposome formulations
according to the invention contain an unencapsulated portion of
the contrast medium. The encapsulated portion is generally
between 15 and 95% of the total concentration. Those
preparations in which between 30 and 75% is encapsulated are
especially suitable, however. The best results were achieved
with preparations in which 40 to 65% of the contrast medium is
encapsulated. It was possible to show that, surprisingly enough,
the free contrast medium portion produces a positive influence of
the diagnostic quality of the preparations according to the
invention.
For the production of formulations according to the
invention, suitable hydrophilic contrast media (diagnostic
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agents) are generally known from radiological practice (e.g., CT,
MRT, nuclear diagnosis). These include, for one thing, the x-ray
contrast media such as, for example, amidotrizoate, metrizoate,
iopromide, iohexol, iopamidol, iosimide, ioversol, iomeprol,
iopentol, ioxilan, iobitridol, ioxaglat, iotrolan, iodixanol,
bis-t{3-N-(2,3-dihydroxypropyl-carbamoyl)-5-carbamoyl}-2,4,6-
triiodo-N-(2,3-dihydroxypropyl)-anilide]-malonic acid and 5-
hydroxyacetylamino-2,4,6-triiodo-isophthalic acid-[(2,3-
dihydroxy-N-methyl-propyl)-(2,3-dihydroxypropyl)]diamide, which
is used in CT. Nonlimiting examples from the area of MRT (NMR)
contrast media are, for example, Gd-DTPA, Gd-EOB-DTPA, Gd-DOTA,
Gd-BOPTA and Mn-DPDP. In the case of MRT contrast media,
compounds based on metal-containing macrocycles, such as, for
example, gadobutrol, are especially suitable for the production
of formulations according to the invention.
In the case of MRT contrast media, those substances that
contain central atoms other than gadolinium can also be used.
Other suitable lanthanides are thus, for example, dysprosium or
ytterbium. In the case of special applications according to the
invention, such substances (such as, e.g., Dy or Yb-EOB-DTPA) can
also be used as opacifying components for computer tomography.
The aqueous phase can also contain the adjuvants that are
known to one skilled in the art, such as, for example, buffer
substances, isotonizing additives or preservative additives.
The lipid components that are used in the formulations
according to the invention are generally described in the
literature. Phospholipids are natural or synthetic phospholipids
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such as, for example, phosphatidylcholine,
phosphatidylethanolamine or sphingolipids, whereby naturally
occurring phospholipids, such as, e.g., soy phosphatidylcholine
(SPC) and egg phosphatidylcholine (EPC) are preferredJ Mixtures
of the above-mentioned components can also be used.
As amphiphilic substances, for example, hexadecyl-
poly(3)glycerol, dialkylpoly(7)glycerol-ether and alkylglucosides
can be mentioned. Mixtures of the above-mentioned components can
also be used. Moreover, other amphiphilic substances that are
obtained synthetically or biotechnologically can also be used,
however, for the production of liposomes according to the
invention. When using amphiphilic substances, so-called
niosomes, i.e., liposomes made of non-ionogeneic vesicle formers,
can be obtained.
As a sterol, especially cholesterol is used.
As charge carriers, for example, components such as fatty
acids (e.g., stearic acid, palmitic acid), dicetylphosphate,
cholesterol hemisuccinate or natural or synthetic phospholipids
such as phosphatidylglycerol, phosphatidylserine, phosphatidic
acid or phosphatidylinositol are used. In addition, charged
amphiphilic substances (see above) can also be used as charge
carriers. Mixtures of the above-mentioned components can also be
used.
In addition, the liposome membrane can also contain
preservative additives, such as, for example, ~-tocopherol as an
antioxidant.
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12
The.liposome-preparations according to the invention do not
contain any surface-hydrophilizing additives, such as, for
example, DSPE-PEG or GM1 (see above) for prolonging the blood
half-life. It was possible to show that DSPE-PEG-containing
preparations have a reduced, acute compatibility compared to
unmodified liposomes.
Especially suitable formulations are obtained if the lipids
that are used as starting substances are present in the following
mixture ratios:
a) 60% soy phosphatidylcholine, 30% cholesterol,
10% soy phosphatidylglycerol,
b) 70% soy phosphatidylcholine, 20% cholesterol,
10% soy phosphatidylglycerol,
c) 75% soy phosphatidylcholine, 20% cholesterol,
5% soy phosphatidylglycerol,
d) 50% soy phosphatidylcholine, 40% cholesterol,
10% soy phosphatidylglycerol,
e) 60% soy phosphatidylcholine, 30% cholesterol,
10% distearoyl phosphatidylglycerol,
f) 70% soy phosphatidylcholine, 20% cholesterol,
10% distearoyl phosphatidylglycerol,
g) 60% soy phosphatidylcholine, 30% cholesterol,
10% dimyristoylphosphatidylglycerol,
h) 60% soy phosphatidylcholine, 30% cholesterol,
10% distearoyl phosphatidic acid,
i) 70% soy phosphatidylcholine, 20% cholesterol,
10% distearoyl phosphatidic acid or
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13
k) 75% soy phosphatidylcholine, 20% cholesterol,
5% distearoyl phosphatidic acid.
The average diameter of liposome formulations according to
the invention is between 100 and 400 nm (measured by photon
correlation spectroscopy (PCS), see Examples). In especially
suitable preparations, the liposomes have an average diameter of
between 150 and 250 nm.
The liposome formulations according to the invention are
stable stored in a refrigerator over a period of at least 9
months, but for the most part of more than 12 months. In
especially suitable cases, corresponding formulations are also
stable at room temperature over this period. Moreover, the
liposome formulations according to the invention can be heat-
sterilized. Tests with formulations according to the invention,
which were treated for 20 minutes at 121~C, thus showed that no
significant alterations occurred.
In the case of the special pharmacological properties of
formulations according to the invention, the limited plasma
stability (e.g., in human plasma) can be mentioned, for one
thing. Thus, it was shown in vitro that the degree of
encapsulation decreased even in the first 2 hours by about 20 to
30%. Up to 6 hours after the liposome suspension was mixed with
the plasma, the portion of encapsulated contrast medium further
decreased (to about 60%). The plasma stability of the
formulations according to the invention in human plasma after 2
hours is preferably in the range of 50-90 or 60-80% of the
originally encapsulated portion.
CA 022l2l62 l997-08-0
14
By the early leakage of contrast medium from the liposomes,
quick elimination of the opacifying components is made possible.
If corresponding empty liposomes are concentrated at later times
in organs of the MPS, the contrast medium does not lead to any
intracellular burden.~ To this extent, generally also no
liposomes with lipophilic contrast media are used within the
framework of the applications according to the invention, since
the latter together with the liposome shell were concentrated at
later times in the liver and spleen.
The maximum concentration of contrast media of liposome
formulations according to the invention in the liver and spleen
within a period of 24 hours is generally less than 10%, but
always less than 20%. At later times after the administration of
formulations according to the invention, no further increase of
the contrast medium concentration in the liver and spleen takes
place compared to early times, i.e., no late concentration
occurs. Despite the low concentration of formulations according
to the invention in the liver and spleen, preferably those
contrast media are encapsulated that have a quick and complete
elimination from the MPS and form, moreover, no toxic
decomposition products.
With the liposome preparations according to the invention,
blood concentrations of up to 75%, generally 30-55%, of the
administered dose are found in the blood at early times (15 to 60
minutes p.i.). After 4 hours, the average blood concentration,
however, is less than 25%, generally approximately 15 to 20%.
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The blood half-lives of the liposomal contrast media
according to the invention are generally less than 8 hours, but
always less than 16 hours.
It was possible to show that the liposome formulations
according to the invention are suitable, surprisingly enough,
especially for use in blood-pool imaging indications. Thus, for
example, iopromide-containing liposomes at a dose of 200 gm of
total iodine/kg in rabbits showed a considerable increase of x-
ray opacity in the blood over the entire study period of 20
minutes. In direct comparison with the aqueous, monomeric
contrast medium iopromide (Ultravist(R)), for example, a
considerably higher difference in contrast (~HU) could be
detected between aorta and liver tissue for the liposomes.
In organ distribution studies of rats, a blood concentration
of about 1.8 mg of iodine/g (assumed blood volume of 58 ml/kg)
was also obtained after 15 minutes with a formulation according
to the invention after i.v. administration of 250 mg of total
iodine/kg (iodine/lipid amount used 1:1.5). This value is in the
range of 1.0 to 5.0 mg of iodine/g, preferably 1.5 to 3.0 mg of
iodine/g, which is considered adequate for diagnostic imaging.
At earlier times (< 15 minutes p.i.), considerably higher iodine
concentrations can be reached, which are also advantageous
diagnostically. The latter are, for example, in the range of a
maximum of 10-25 mg of iodine/g or 15-20 mg of iodine/g.
Despite the relatively high contrast medium concentrations,
which are required in this CT application, here, surprisingly
enough, the formulations according to the invention with their
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16
relatively low iodine/lipid ratios are especially suitable. The
ratio of included iodine to lipid used in the formulations
according to the invention is thus only in the range of about 0.1
to 1.4, preferably 0.2-0.8 mg, especially preferably 0.25-0.65 mg
of encapsulated iodine/mg of lipid.
With a view to ensuring the use of formulations according to
the invention in MRT, it was also possible to reach an adequate
concentration of opacifying components over a diagnostically
relevant period based on organ-distribution studies of rats.
Thus, for example, after i.v. administration of 0.3 mmol of total
Gd/kg (liposomal Gd-EOB-DTPA, Gd/lipid amount used [~mol/mg] =
1:1.5) 15 minutes p.i., a blood concentration of about 1.7 ~mol
of Gd/g (assumed blood volume of 58 ml/kg) was obtained. After
60 minutes, the blood concentration was still approximately 1.1
~mol of Gd/g and thus still in the diagnostically relevant
concentration range of 0.15-2.5 ~mol/g or preferably 0.5 to 2.0
~mol/g. Similar to the above-described application in CT,
basically higher contrast medium concentrations in the blood can
in turn result here at early times (< 15 minutes).
For the production of formulations according to the
invention for MRT, mainly macrocyclic contrast media, such as
gadobutrol, are especially suitable. Corresponding formulations
ensure quick and complete elimination of the contrast medium.
The formulations according to the invention are distinguished,
moreover, by a relaxivity that is not altered or is only slightly
altered compared to the free contrast medium.
17
Embodiments:
The following examples are to explain the object of the
invention without intending that they be limited to this object.
The abbreviations that are used in this case are defined
below:
FEA: X-ray fluorescence spectroscopy (Kaufman, L. et
al., Invest. Radiol. 11, 210-215 (1976))
PCS: Photon correlation spectroscopy, process for
measuring particle sizes of less than 1 ~m
(device: Nicomp model 370)
0 average diameter (determined by PCS)
SPC: soy phosphatidylcholine, lipoid S 100, the company
Lipoid KG, Ludwigshafen
SPG: soy phosphatidylglycerol, lipoid SPG, the company
Lipoid KG
CH: cholesterol, powdered cholesterol, Merck company,
Darmstadt
CHHS chloride hemisuccinate, Sigma company, Deisenhofen
DSPG distearoyl phosphatidylglycerol, Sygena company,
Liestal, CH
DSPS distearoyl phosphatidylserine, Sygena company,
Liestal, CH
DSPA distearoyl phosphatidic acid, Sygena company,
Liestal, CH
DPPA dipalmitoyl phosphatidic acid, Sygena company,
Liestal, CH
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18
DMPG dimyristoyl phosphatidylglycerol, Sygena company,
Liestal, CH
SPC35 partially hydrogenated soy phosphatidylcholine;
lipoid SPC 35, the company Lipoid KG, Ludwigshafen
EPC egg phosphatidylcholine; Lipoid E 100, the company
Lipoid KG, Ludwigshafen
MW + SD: mean value + standard deviation
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19
Example 1: Production of Iopromide-Containing Liposome
Suspensions
Iopromide-containing liposomes with various lipid
compositions are produced with a continuous high-pressure
extrusion method (incl. freeze-thaw) (Schneider, T. et al., Drug.
Dev. Ind. Pharm. 20, 2787-2807 (1994~) and studied with respect
to their properties.
Lipid 0 Iodine Encapsula- Osmolality
Compositiontnm] Content tion tmOsm/kg]
[mg/g] l%]
SPC/CH/SPG239+30 90.0+4.8 41.2+2.2 303+31
6:3:1
(n = 6)
SPC/CH/SPG 253 73.0 44.4 279
5:3:2
SPC/CH/SPG 157 84.8 44.3 273
5:4:1
SPC/CH/CHHS 210 80.6 31.1 271
4:5:1
SPC/CH/SPG 162 74.9 32.0 263
5:4:1
SPC/CH/SPG 217 88.2 45.5 337
7:2:1
SPC/CH/DSPG255+1196.4+0.6 46.1+1.0 468+59
6:3:1
(n = 2)
SPC/CH/DPPA 283 95.8 55.1 330
6:3:1
SPC/CH/DSPS 294 81.8 19.3# 478
7.8:2:0.2
SPC/CH/DMPG 282 103.5 57.0
6:3:1
SPC35/CH/SPG209 100.8 37.9
6:3:1
EPC/CH/SPG 347 90.9 60.9 387
6:3:1
Iodine/lipid amount used = 1:1.5
iodine/lipid amount used = 1:1
# extrusion at room temperature
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Example 2: Shelf Life of Iopromide-Containing Liposome
Suspensions
Liposomes that are produced according to Example 1
(SPC/CH/SPG 6:3:1, iodine/lipid amount used l:l.S) are stored in
a refrigerator or at room temperature and studied with respect to
their stability after 9 months.
Time 0 Iodine Content Encapsulation
tnm] tmg/g] t~]
t = 0 264 97.5 38.7
t = 9 months 217 97.6 40.0
4-8~C
t = 9 months 229 98.1 40.0
room
temperature
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Example 3: Production of Gd-containing Liposome 8uspensions
Liposomes with Gd-containing MRT contrast media are produced
using the high-pressure extrusion process that is described under
Example 1.
Lipid Contrast 0 Gd Content Encapsu- Osmo-
Composi- Medium tnm] l~mol/ml] lation lality
tion t%] tmOsm/kg]
SPC/CH Gd-EOB- 359 110.7 51.3 364
7:3 DTPA
SPC/CH " 105 110.7 32.6 381
7:3
SPC/CH/Gadobu- 227 80.2 41.0 not
SPG trol determined
6:3:1
SPC/CH/ " 141 80.6 49.5 138
SPG
6:3:1
SPC/CH/ " 210+7110.8+2.357.4+1.5 206+5
SPG#
7:2:1
iodine/lipid amount used = 1:1.5
# n = 3, all others n = 1
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Example 4: Plasma Stability of Iopromide-Containing Liposomes
Liposome suspensions that are produced according to Example
1 (batch A: 198 nm, 42.9% encapsulated, batch B: 103 nm, 34.7%
encapsulated) were mixed with human plasma, whereby an iodine
concentration of about 5 mg/ml was set. In each case, 1 ml of
this plasma-contrast medium mixture was then dialyzed in a
Dianorm equilibrium dialysis apparatus (Dianorm, Heidelberg)
compared to the corresponding human plasma by dialysis membranes
with a cutoff of 5000 Da (Dianorm). At various times, samples
were taken from the retentate and permeate side, and the iodine
content was determined using x-ray fluorescence spectroscopy
(FEA). The results obtained can be seen in Figure 1.
23
~xample 5: Organ Distribution of Iopromide-Containing Liposomes
in Rats
The liposome suspension that is presented in Example 4
(batch A) was injected at a dose of 250 mg of total iodine/kg
into 16 male rats (weight: 137-160 g), and in each case, 4
animals were killed 0.25; l; 4 and 24 hours after injection.
Then, the liver, spleen, lung and blood were checked for their
iodine content using FEA. The results (% of the administered
dose/organ) can be seen in the table below.
Time 0.25 1 4 24
p. i. lh]
Organ (MW+SD) (MW+sD) (MW+sD) (MW+SD)
Blood 41.3+4.8 31.0+5.0 12.5+3.2 2.1+1.3
Lung 0.8+0.1 0.6+0.1 0.4+0.1 0.2+0.1
Liver 5.1+0.7 4.8+1.1 5.3+0.2 2.9+0.9
Spleen 1.7+0.2 2.7+0.1 3.S+0.4 1.9+0.4
CA 02212162 1997-08-0
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Example 6: Organ Distribution of Surface-Modified (DSPE-PEG)
Liposomes
A DSPE-PEG-containing liposome suspension (SPC/CH/SPG 6:3:1
+ 5 mol% of DSPE-PEG 2000-204 nm, 45~ encapsulated) was injected
at a dose of 250 mg of total iodine/kg into 16 male rats (weight:
136-160 g), and in each case 4 animals were killed 0.25; 1; 4 and
24 hours after injection. Then, the liver, spleen, lung and
blood were checked for their iodine content using FEA. The
results (% of the administered dose/organ) can be seen in the
table below.
Time 0.25 1 4 24
p. i. th]
Organ (MW+SD) (MW+SD) (MW+SD) (MW+SD)
Blood 37.9+2.6 29.7+3.3 27.7+0.6 8.6+3.5
Lung 0.6+0.0 0.4+0.1 0.4+0.1 0.4+0.1
Liver 4.5+0.4 3.4+0.5 3.8+0.3 4.1+0.7
Spleen 0.6+0.1 1.3+0.3 2.2+0.8 2.2+0.9
CA 02212162 1997-08-0
Example 7: Blood-Pool Imaging (CT) in Rabbits
The liposome suspension that is presented in Example 4
(batch A) was examined at a dose of 200 mg of total iodine/kg in
rabbits. As a control, the monomeric x-ray contrast medium
Ultravist(R) (Iopromide (INN)) was used. The x-ray opacity was
measured in Hounsfield Units (HU) in the aorta and in the liver
tissue from 0 to 20 minutes after one-time intravenous
administration (spiral-CT, somatom plus S, Siemens company, at
120 kV). From the data, the surface below the signal-time curve
for the aorta and for the liver was calculated over 20 minutes in
HU min; the difference between aorta and liver tissue is a
yardstick for contrast quality. The results were depicted in
Figure 2.
As is shown by Figure 2, the signal difference between aorta
and liver for the iopromide liposomes is considerably higher than
for the free iopromide (Ultravist(R)).
CA 02212162 1997-08-05
26
Example 8: Plasma Stability of Gd-DTPA-EOB-containing Liposomes
The stability of Gd-EOB-DTPA-containing liposomes (batch A:
359 nm, 51.3% encapsulated, batch B: 105 nm, 32 . 6% encapsulated,
see also Example 3 ) in human plasma was examined as described
under Example 4. In this case, the Gd concentration that was
used in GGD was approximately 20 ~mol of Gd/ml. The result was
depicted in Figure 3.
CA 02212162 1997-08-0~
Example 9: Blood Level Plots after Administration of Gd-EOB-
DTPA-containing Liposomes in Rats
The liposome suspensions that are presented in Example 8
(batches A and B) were injected at a dose of 0.3 mmol of total
Gd/kg into 16 male rats (weight: 137-158 g), and in each case, 4
animals were killed 0.25; 1; 4 and 24 hours after injection.
Then, the Gd content in the blood was determined using ICP-AES
(inductively coupled plasma atom emission spectroscopy).
Visualization of the liver concentrations was unnecessary here,
since the unencapsulated Gd-EOB-DTPA also accumulated
specifically in the liver (liver contrast media for MRT). The
results (% of the administered dose/blood) can be seen in the
table below.
Time p.i. (hours)0.25 1 4 24
(MW+SD) (MW+SD) (MW+SD) (MW+SD)
Batch A 35.9+0.6 21.5+1.2 10.2+2.3 2.1+0.5
Batch B 19.3+2.5 17.8+0.9 11.4+2.9 2.6+1.0
CA 02212162 1997-08-0
28
Example 10: Blood-pool Enhancement (CT) by Gd-EOB-DTPA Liposomeq
in Rabbits
A Gd-EOB-DTPA-containing liposome suspension (batch A, see
Example 8 ) was administered at a dose of 0.3 mmol of Gd/kg i.v.
(lateral ear vein) to an anesthetized rabbit (3 ml/min). Since
Gd-EOB-DTPA, which is used actually as an MRT liver contrast
medium, also absorbs x rays, it was possible to use, as a trial,
computer tomography (CT) to detect the concentration of the
liposomal components in the blood. Figure 4 depicts the plot of
x-ray opacity in ~HU in the aorta over a period of one hour.
