CA2212162A1 - Liposomes that contain contrast media for the visualization of intravascular space - Google Patents

Abstract

The invention relates to liposomal contrast medium preparations for diagnostic visualization of intravascular space as well as their use in imaging diagnosis.

Description

CA 02212162 1997-08-0~

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 (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 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.

CA 02212162 1997-08-0~

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 CA 02212162 1997-08-0~

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 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 CA 02212162 1997-08-0~

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 CA 02212162 1997-08-0~

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 CA 02212162 1997-08-0~

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 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 CA 02212162 1997-08-0~

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.

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 CA 022l2l62 l997-08-0 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 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 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.

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

CA 022l2l62 l997-08-05 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 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 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 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 CA 02212162 1997-08-0~

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.

~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 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 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)).

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 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.

Claims (29)

Claims

1. Active ingredient-containing liposome formulation, 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 c) the active ingredient is an x-ray or MRI contrast medium or a radiodiagnostic agent.

2. Active ingredient-containing liposome formulation, wherein 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.

3. Active ingredient-containing liposome formulation, wherein 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.

4. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein phosphatidylcholine, phosphatidylethanolamine, soy phosphatidylcholine, egg phosphatidylcholine or a sphingolipid is used as a phospholipid.

5. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein hexadecylpoly(3)glycerol, dialkylpoly(7)glycerol ether or an alkyl glucoside is used as an amphiphilic substance.

6. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein cholesterol is used as a sterol.

7. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein a fatty acid, dicetylphosphate, cholesterol hemisuccinate, phosphatidylglycerol, phosphatidylserine, phosphatidic acid or phosphatidylinositol is used as a charge carrier.

8. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein at least one amphiphilic substance is used as a charge carrier.

9. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein the liposomes have an average diameter of 150-250 nm.

10. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein the active ingredient is encapsulated only partially in the liposomes.

11. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein 30-75% of the active ingredient is encapsulated in the liposomes.

12. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein 40-65% of the active ingredient is encapsulated in the liposomes.

13. Active ingredient-containing liposome formulation according to claim 1, wherein the following mixture ratios of the components are present:
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 k) 75% soy phosphatidylcholine, 20% cholesterol, 5% distearoyl phosphatidic acid.

14. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein the active ingredient is amidotrizoate, metrizoate, iopromide, iohexol, iopamidol, iosimide, ioversol, iomeprol, iopentol, ioxilan, iobitridol, ioxaglat, iotrolan, iodixanol, bis-[{3-N-(2,3-dihydroxypropyl-carbamoyl)-5-carbamoyl}-2,4,6-triiodo-N-(2,3-dihydroxypropyl)-anilide]-malonic acid or 5-hydroxyacetylamino-2,4,6-triiodo-isophthalic acid-[(2,3-dihydroxy-N-methyl-propyl)-(2,3-dihydroxypropyl)]diamide.

15. Active ingredient-containing liposome formulation according to one of claims 1-3, wherein the active ingredient is Gd-DTPA, Gd-EOB-DTPA, Yb-EOB-DTPA, Dy-EOB-DTPA, Gd-DOTA, Gd-BOPTA, gadobutrol or Mn-DPDP.

16. Use of an active ingredient-containing liposome formulation for the production of an agent for diagnostic radiology, wherein 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 c) the active ingredient is an x-ray contrast medium.

17. Use of an active ingredient-containing liposome formulation for the production of an agent for diagnostic radiology, wherein 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 in an x-ray contrast medium.

18. Use of an active ingredient-containing liposome formulation for the production of an agent for diagnostic radiology, wherein a) the following mixture ration 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 contrast medium.

19. Use of an active ingredient-containing liposome formulation according to one of claims 16-18 for the production of an agent for the visualization of intravascular space by diagnostic radiology.

20. Use of an active ingredient-containing liposome formulation according to one of claims 16-18 for the production of an x-ray contrast medium for computer tomography.

21. Use of an active ingredient-containing liposome formulation according to claim 20 for the production of an agent for visualization of intravascular space by diagnostic radiology using computer tomography.

22. Use of an active ingredient-containing liposome formulation for the production of an agent for MRI diagnosis, wherein 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 c) the active ingredient is an MRI contrast medium.

23. Use of an active ingredient-containing liposome formulation for the production of an agent for MRI diagnosis, wherein 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 MRI contrast medium.

24. Use of an active ingredient-containing liposome formulation for the production of an agent for MRI diagnosis, wherein 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 MRI contrast medium.

25. Use of an active ingredient-containing liposome formulation according to one of claims 22-24 for the production of an agent for the diagnostic visualization of intravascular space using MRI.

26. Use of an active ingredient-containing liposome formulation for the production of an agent for radiodiagnosis, wherein 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 c) the active ingredient is a radiodiagnostic agent.

27. Use of an active ingredient-containing liposome formulation for the production of an agent for radiodiagnosis, wherein 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 a radiodiagnostic agent.

28. Use of an active ingredient-containing liposome formulation for the production of an agent for radiodiagnosis, wherein 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 a radiodiagnostic agent.

29. Use of an active ingredient-containing liposome formulation according to one of claims 26-28 for the production of an agent for the radiodiagnostic visualization of intravascular space.