EP0806969A2

EP0806969A2 - Liposomes containing contrast media for blood pool imaging

Info

Publication number: EP0806969A2
Application number: EP96904033A
Authority: EP
Inventors: Andreas Sachse; Georg Rössling; Jens Leike; Werner Krause; Gabriele Schuhmann-Giampieri; Peter Muschick; Wolf-Rüdiger Press
Original assignee: Schering AG
Current assignee: SACHSE Andreas
Priority date: 1995-02-09
Filing date: 1996-02-09
Publication date: 1997-11-19
Also published as: AU4789096A; NO973666D0; WO1996024381A3; JPH10513466A; NO973666L; WO1996024381A2; DE19606326A1; CA2212162A1

Abstract

Liposome preparations containing contrast media for diagnostic blood pool imaging, and use thereof in diagnostic imaging. The invention thus concerns liposome formulations containing active substances and characterized in that: (a) the following proportions of lipids in the mixture: 40-90 % phospholipids or amphiphilics, 10-50 % sterols, 0-25 % charge carriers; (b) the liposomes have a mean diameter of 100-400 nm; (c) the active substance is an X-ray or MRI contrast agent, or a radiodiagnostic agent.

Description

Contrast agent-containing liposomes

for the representation of the intravascular space

The invention relates to liposome formulations containing ontrast with a long intravascular residence time, which are suitable for the representation of the intravascular space.

State of the art

Liposomes have become increasingly important in recent years as potential carrier systems for the various types of contrast media. The use of liposomal contrast medium formulations has been described for all imaging diagnostic methods (X-ray diagnostics, computer tomography, MRI diagnostics, radio diagnostics) (Seltzer, St. E., Liposomes in diagnostic imaging. In: Gregoriadis, G. (ed.), Liposomes as drug carriers, John Wiley & Sons Ltd., Chichester, New York, Brisbane, Toronto, Singapore 1988, p. 509).

Due to their structure, the liposomes enable the inclusion of hydrophilic contrast agents in the water phase as well as the inclusion of lipophilic contrast agents in the bilayer phase (Seltzer, St. E., Radiology 171, 19-21 (1989)). After intravenous administration, liposomes are preferentially enriched in αen organs of the mononuclear phagocyte system (MPS, also called RES), the highest concentrations being reached in the liver and spleen. This so-called passive targeting is used in the case of contrast medium-containing liposomes in order to achieve a selective enrichment of these substances in the healthy liver. For example, the liposo al-encapsulated X-ray contrast agent Iopro id (Ultravist ^® ) was able to differentiate tumorous changes in the liver in the rabbit model (Sachse, A. et al., Invest. Radiol. 28, 838-844 (1993);

The in vivo behavior of liposomes is high. Influence measurement by changing the chemical composition and the physical properties of vesicles. The blood half-life and organ distribution of the liposomes are influenced by parameters such as vesicle size, surface charge (Ze potential), lipid composition and lipid dose (Senior, JH, CRC Cπt. Rev. Therap. Drug Camer Syst. 3, 123-193 (1987) ). For example, the blood half-life of liposomes can be reduced by reducing the liposome size or by rigidifying the membrane e.g. can be significantly extended by using saturated phospholipids (e.g. distearoylphosphatidylcholm, DSPC). The introduction of a load, in turn, can lead to a drastic increase in MPS intake and thus a reduction in the blood half-life.

In addition, the blood retention time is very strongly influenced by the applied lipid dose or number of particles. At higher lipid concentrations, there is a decrease in the relative liver uptake, probably due to a saturation of the liver uptake mechanism. At the same time, a higher spleen uptake and a prolonged blood retention period are observed.

With regard to therapeutic or diagnostic applications of liposomes in organs outside the MPS, there was great interest in significantly increasing the blood half-life of liposomes. This was hoped for, among other things to be able to achieve increased extravasation of corresponding liposomes in areas with damaged vascular endothelium (eg tumors or inflammation). In recent years it has been shown that the surface modification of liposomes (hydrophilization) can change their blood retention time. Lipid derivatives of polyethylene glycol (eg DSPE-PEG1900) have proven to be particularly advantageous compared to the polysaccharides or glycolipids (eg GM1) initially used (Allen, TM, Adv. Drug. Delivery Rev. 13, 285-309 (1994)). This effect of PEGylation is attributed to the formation of a steric barrier on the surface of the liposome, by means of which the interaction of the liposomes with various plasma components (eg plasma proteins or opsonins) is significantly changed. The blood half-lives of such sterically stabilized liposomes (SSL) when using PEGs with molecular weights between 1900 and 5000 are in the range of approximately 9 to 16 hours. The liver uptake of corresponding liposomes (100 - 200 nm) reached values up to slightly below 25% of the applied dose. It should be noted, however, that the liver and spleen are still the main organs for receiving SSL.

If liposeal preparations are used for imaging the intravascular space (blood pool imaging), it is necessary to largely avoid the liposomes being absorbed into the MPS. Because of the relatively large blood volume, high contrast medium concentrations are required in the vascular system, particularly in CT, in order to enable meaningful imaging. If there is an accumulation of the liposomal contrast medium, for example in the liver and spleen, as part of diagnostic examinations in the area of the vascular system, this can u. U. lead to an adverse impact on the functions of the MPS (eg immune defense). In mice, small to medium doses of placebo liposomes (20 - 80 mg / kg) were able to demonstrate a significant impairment of the MPS absorption capacity for carbon particles (Allen, TM et al., Journ. Pharm. Exper. Therap. 229 , 1984, 267-275). About that In addition, the encapsulated contrast medium can also lead to changes in the RES.

As already mentioned above, liposomal contrast media have already found their way into the development of organ-specific contrast media. For example, WO 88/09165 describes sprayable, aqueous liposome preparations with iodine-containing X-ray contrast agents and a method for producing corresponding formulations. Due to the size (0.15 - 3 µm) and the high contrast medium (iodine / lipid ratio between 1.5 and 6 g / g), appropriate preparations should be particularly suitable for the representation of the liver.

EP 0160552 A2 describes micellar or liposomal contrast media for magnetic resonance tomography (MRT, MRI). According to the invention, the small umlamellar liposomes (SUV 60 ± 10 nm) Application to tumor-bearing mice lead to an increased tumor accumulation of the liposomal Gd-DTPA.

WO 90/04943 describes liposomal MRI contrast agents, methods for their preparation and applications. The liposomes according to the invention have an average diameter below 50 nm and, in addition to being used for imaging tumors of the liver and spleen, should also be suitable for imaging the vascular system, the heart and the perfusion of tissues (blood pool imaging) . However, these small liposomes have the disadvantage that, because of their restricted volume, only small amounts of hydrophilic components can be included. Furthermore, depending on the lipid composition of the liposome membrane, a significant reduction in the relaxivity of the encapsulated component was described for MRI contrast media. Recently lipophilic, paramagnetic chelates have therefore become increasingly important. These components are built into the liposome membrane (bilayer) and therefore behave like the lipid membrane with regard to their pharmacokmetic. Corresponding liposomes (Memsomes), which extend the Blood half-life additionally surface modified (PEGylilert) are said to be particularly suitable for blood pool imaging due to their high relaxivity (Tilcock, T., J. Liposome Res. 4, 909-936 (1994)).

The latter author also describes surface-modified (PEG) liposomes for imaging the vascular system in nuclear diagnostics. The radioactive component can be included either in the inner water phase or in the membrane phase. Radiolabelled liposomes (PE-DTTA and 99mTc) with a mean diameter of approx. 100 nm showed a blood half-life of more than 12 hours with surface hydrophilization with 4 - 6 mol ° ό PΞ-PEG 6000 (SSL) . After 8 hours, with appropriate preparations, high activity in the heart and blood vessels could be obtained. At the same time, however, a clear accumulation of liver was detectable.

The visualization of the vascular system is very important in radiological practice. New contrast media for a specific visualization of vessels and the heart (e.g. particulate systems, macromolecules) should remain in the vascular system for a longer period after an intravenous injection. This "blood pool effect" of new contrast media could cause many pathological conditions, on the one hand due to a reduction in blood flow (e.g. due to thrombosis, embolism, tumors) or on the other hand due to an abnormal increase in blood flow (e.g. due to a disturbance in capillary integrity) are characterized, can be diagnosed more precisely with non-invasive methods. Furthermore, an exact representation of the perfusion of different tissues and organs (e.g. heart, lungs) or of pathological changes in the heart (e.g. heart valve defects) could be achieved.

The previous approaches to providing corresponding contrast agents in CT and MRI failed due to the inadequate, pharmaceutical or pharmacological properties of these drug carrier systems. That's the way it is necessary to be able to reproducibly produce corresponding contrast media with defined chemical-physical properties in large quantities. In addition, despite the need to administer large amounts of contrast medium (especially for use in CT), the corresponding carrier systems must be very well tolerated in order to arrive at a positive risk / benefit assessment. Since the diagnostically relevant examination period for the presentation of the intravascular space is limited to approximately two hours after the injection (pi), appropriate contrast agents should be excreted as quickly and completely as possible. Furthermore, there should be no excessive accumulation in areas not diagnostically relevant for this indication.

There is therefore a need for liposomal contrast media for the representation of the intravascular space, which the above-mentioned. Avoid disadvantages, have an adequate blood half-life and accumulate only to a small extent in the liver, spleen or other organs. This object has been achieved by the present invention, in particular by the liposomal contrast agent formulations as characterized in the claims.

The invention therefore relates to active substance-containing liposome formulations, characterized in that a) the following mixing ratio of the 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 substance is an X-ray or MRI contrast medium or a radio diagnostic. The invention preferably relates to active substance-containing liposome formulations, characterized in that a) the following mixing 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 substance is an X-ray or MRI contrast medium or a radio diagnostic agent.

The invention particularly preferably relates to liposome formulations containing active ingredient, characterized in that a) the following mixing ratio of the lipids is present: 60-70% phosphatidylcholm, 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 substance is an X-ray or MRI contrast agent or a radio diagnostic agent.

The processes or process steps suitable for the production of contrast agent-containing liposome preparations according to the invention can generally the standard methods known in liposome technology. (e.g. New, R.R.C., Preparation of liposomes. In: New, R.R.C. (ed.), Liposomes: a practical approach, Oxford University Press, New York, 1990). However, continuous high-pressure extrusion is particularly suitable for producing liposome suspensions with the properties according to the invention

(WO 94/08626). In addition, however, other mechanical or multi-phase dispersion processes can also be used, for example, to produce preparations according to the invention. The liposome preparations according to the invention produced in this way can be stored directly or in lyophilized form or kept ready for use become. The latter samples must be resuspended before use.

The liposome formulations according to the invention contain, in addition to the encapsulated portion of a hydrophilic (water-soluble) contrast medium, an unencapsulated portion of the same. The encapsulated portion is usually between 15 and 95% of the total concentration. However, preparations in which between 30 and 75% are encapsulated are particularly suitable. The best results were achieved with preparations in which 40 to 65% of the contrast medium are encapsulated. It could be shown that, surprisingly, the free contrast agent component has a positive influence on the diagnostic quality of the preparations according to the invention.

Hydrophilic contrast media (diagnostics) suitable for producing formulations according to the invention are generally known from radiological practice (e.g. CT, MRI, nuclear diagnostics). These include the X-ray contrast agents such as amidotrizoate, metrizoate, iopromide, iohexol, iopamidol, iosimide, ioversol, iomeprol, iopentol, ioxilan, iobitridol, ioxaglate, iotrolan, iodixanol, bis- [{3-D- (2, hydroxyl -carbamoyl) -5- carbamoyl} -2, 4, 6-trιιod-N- (2, 3-dιhydroxypropyl) anilide] - malonic acid and 5-hydroxyacetylammo-2, 4, 6-trιιod- isophthalic acid- [(2, 3-di-hydroxy-N-methyl-propyl) - (2,3-dihydroxypropyl)] diamide, which are used in CT. Non-limiting examples from the field of MRT (NMR) contrast media are, for example, Gd-DTPA, Gd-EOB-DTPA, Gd-DOTA, Gd-BOPTA and Mn-DPDP. In the case of MRI contrast media, compounds based on metal-containing macrocycles, such as, for example, gadobutrol, are particularly suitable for producing formulations according to the invention.

Substances containing central atoms other than gadolinium can also be used in MRI contrast media. Other suitable lanthanides are, for example Dysprosium or ytterbium. In special applications according to the invention, such substances (such as, for example, Dy- or Yb-EOB-DTPA) can also be used as contrast-imparting components for computer tomography.

The aqueous phase can also contain the auxiliaries known to the person skilled in the art, such as, for example, buffer substances, isotonizing additives or preserving additives.

The used in the formulations according to the invention

Lipid components are generally described in the literature. The phospholipids are natural or synthetic phospholipids such as phosphatidylcholine, phosphatidylethanolamine or sphingolipids, with naturally occurring phospholipids such as e.g.

Soy phosphatidylcholine (SPC) and egg phosphatidylcholine (EPC) are preferred. Mixtures of the above components can also be used.

Examples of amphiphilic substances are hexadecyl poly (3) glycerol, dialkyl poly (7) glycerol ether and alkyl glucosides. Mixtures of the above components can also be used. In addition, however, other synthetically or biotechnologically obtained amphiphilic substances can also be used for the production of liposomes according to the invention. When using amphiphilic substances, so-called niosomes, i.e. Liposomes obtained from non-ionogenic vesicle formers.

Cholesterol in particular is used as the sterol.

Components such as fatty acids (e.g. stearic acid, palmitic acid), dicetyl phosphate, cholesterol isuccinate or natural or synthetic phospholipids such as phosphatidylglycerol, phosphatidylserine, for example, are used as charge carriers.

Phosphatidic acid or phosphatidylinositol used. Furthermore, charged amphiphilic substances (see above) can also be used as charge carriers. Mixtures of the above components can also be used. In addition, the liposome membrane can also contain preservative additives such as tocopherol as an antioxidant.

The liposome preparations according to the invention do not contain any surface-hydrophilizing additives such as DSPE-PEG or GM1 (see above) to prolong the blood half-life. It could be shown that preparations containing DSPE-PEG have a reduced, acute tolerance to unmodified liposomes.

Particularly suitable formulations are obtained when the lipids used as starting materials are present in the following mixing ratios:

a) 60% soy phosphatidycholine, 30% cholesterol, 10% soy phosphatidylglycerol, b) 70% soy phosphatidycholine, 20% cholesterol, 10% soy phosphatidyl glycerol, c) 75% soy phosphatidycholine, 20% cholesterol,

5% soy phosphatidylglycerol, d) 50% soy phosphatidycholine, 40% cholesterol, 10% soy phosphatidylglycerol, e) 60% soy phosphatidycholine, 30% cholesterol, 10% distearoylphosphatidylglycerol, f) 70% soy phosphatidroglyphyl, 20% chololol, 20% chololol, 20% Soy phosphatidycholine, 30% cholesterol,

10% dimyristoylphosphatidylglycerol, h) 60% soy phosphatidycholine, 30% cholesterol,

10% distearoylphosphatidic acid, i) 70% soy phosphatidycholine, 20% cholesterol, 10% distearoylphosphatidic acid or k) 75% soy phosphatidycholine, 20% cholesterol,

5% distearoylphosphatidic 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 Particularly suitable preparations, the liposomes have average diameters between 150 and 250 nm.

The liposome formulations according to the invention are usually stable for more than 12 months when stored in the refrigerator for a period of at least 9 months. In particularly suitable cases, corresponding formulations are stable over this period even at room temperature. In addition, the liposome formulations according to the invention can be heat sterilized. Experiments with formulations according to the invention which showed 20 min. were treated at 121 ° C that no significant changes occurred.

With the special pharmacological properties of formulations according to the invention, the limited plasma stability (e.g. in human plasma) must be mentioned first of all. It was shown in vitro that the degree of encapsulation decreased by approximately 20 to 30% in the first 2 hours. Up to 6 hours after mixing the liposome suspension with the plasma, the proportion of the encapsulated contrast medium continued to decrease (to approx. 60%). The plasma stability of the formulations according to the invention in human plasma after 2 h is preferably in the range from 50-90 or 60-80% of the originally encapsulated portion.

The early leakage of the contrast agent from the liposomes enables the contrast-giving component to be eliminated quickly. If corresponding, empty liposomes are later accumulated in organs of the MPS, there is no intracellular exposure to the contrast medium. In this respect, as a rule, within the scope of applications according to the invention. also do not use liposomes with lipophilic contrast agents, since these would accumulate in the liver and spleen together with the liposome envelope at later times.

The maximum contrast enhancement of liposome formulations according to the invention in the liver and spleen within a period of 24 hours is generally below 10%, but always below 20%. At later times, no further increase occurs after the formulations according to the invention have been administered the contrast agent concentration in the liver and spleen compared to the early points in time, ie there is no late enrichment. In spite of the low concentration of formulations according to the invention in the liver and spleen, it is preferred to encapsulate those contrast agents which have a rapid and complete elimination from the MPS and, moreover, do not form any toxic decomposition products.

With liposome preparations according to the invention, blood concentrations of up to 75% of the applied dose are found in the blood at an early point in time (15 to 60 min p.i.), generally 30-55%. After 4 hours, however, the mean blood concentration is below 25%, usually 15 to 20%. 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 could be shown that the liposome formulations according to the invention are surprisingly particularly suitable for use in blood pool imaging indications. For example, iopromide-containing liposomes in a dose of 200 mg total iodine / kg in rabbits showed a significant increase in X-ray density in the blood over the entire examination period of 20 minutes. In direct comparison to the aqueous, monomeric contrast agent iopromide (Ultravist ^® ), a significantly higher contrast difference (ΔHU) between the aorta and liver tissue could be detected for the liposomes, for example.

In organ distribution studies in rats, an i.v. Administration of 250 mg total iodine / kg (used iodine / amount of lipid 1: 1.5) also a blood concentration of approx. 1.8 mg iodine / g (assumed

Blood volume 58 ml / kg) obtained after 15 min. This value is in the range of 1.0 to 5.0 mg iodine / g, preferably 1.5 to 3.0 mg iodine / g, which are considered sufficient for diagnostic imaging. At earlier times (<15 min pi), significantly higher iodine concentrations can be reached are also diagnostically advantageous. These are, for example, in the range of a maximum of 10-25 mg iodine / g or 15-20 mg iodine / g.

Despite the relatively high contrast agent concentrations required in this CT application, the formulations according to the invention with their relatively low iodine / lipid quotients are surprisingly particularly suitable here. The ratio of trapped iodine to lipid used in the formulations according to the invention is thus only in the range from about 0.1 to 1.4, preferably 0.2-0.8 mg, particularly preferably 0.25-0.65 mg of encapsulated iodine / mg Lipid.

With regard to the use of formulations according to the invention in MRT, it was also possible to achieve sufficient enrichment of the contrast-giving component over a diagnostically relevant period using organ distribution studies in rats. For example, after i.v. Administration of 0.3 mmol total Gd / kg (liposomal Gd-EOB-DTPA, amount of Gd / lipid used [μmol / mg] = 1: 1.5) for 15 minutes p.i. a blood concentration of approx. 1.7 μmol Gd / g (assumed blood volume 58 ml / kg). After 60 minutes, the blood concentration was still approximately 1.1 μmol Gd / g and thus still in the diagnostically relevant concentration range of 0.15-2.5 μmol / g or preferably 0.5 to 2.0 μmol / g. Similar to the application in CT described above, here too, significantly higher contrast agent concentrations in the blood can result at early points in time (<15 min).

Macrocyclic contrast agents such as Gadobutroi are particularly suitable for producing formulations according to the invention for MRI. Appropriate formulations ensure that the contrast medium is excreted quickly and completely. The formulations according to the invention are furthermore distinguished by a relaxivity which is not or only slightly changed compared to the free contrast medium. Execution example:

The following examples are intended to explain the subject matter of the invention without wishing to restrict it to them.

The abbreviations used here are defined below: FEA: X-ray fluorescence spectroscopy (Kaufman, L. et a., Invest. Radiol. 11, 210-215 (1976))

PCS: Photon Correlation Spectroscopy, Methods for

Measurement of particle sizes below 1 μm (device: Nicomp odel 370) 0 Average diameter (determined by PCS)

SPC: soy phosphatidylcholine, Lipoid S 100, from Lipoid KG, Ludwigshafen

SPG: soy phosphatidylglycerol, Lipoid SPG, Lipoid K_

CH: Cholesterol, powdered cholesterol, Merck, Darmstadt

CHHS cholesterol hemisuccinate, Sigma, Deisenhofen

DSPG Distearoylphosphatidylglycerol, Sygena, Liestal, CH

DSPS distearoylphosphatidylserine, Sygena, Liestal, CH DSPA distearoylphosphatidic acid, Sygena, Liestal, CH

DPPA Dipaimitoylphosphatidic acid, Sygena, Liestal, CH

DMPG Di yristoylphosphatidylglycerol, Sygena, Liestal,

CH SPC35 Partially hydrogenated soy phosphatidycholine; Lipoid SPC 35, from Lipoid KG, Ludwigshafen

EPC egg phosphatidycholine; Lipoid E 100, from Lipoid KG, Ludwigshafen

MW ± SD: mean ± standard deviation

Example 1: Preparation of lopromide-containing liposome suspensions

Using a continuous high-pressure extrusion method (including freeze-thaw) (Schneider, T. et al., Drug. Dev. Ind. Pharm. 20, 2787-2807 (1994)), iopromide-containing liposomes with different lipid compositions are produced and with regard to examined their properties.

Lipidz total iodine content encapsulation osmosis nality [nm] [mg / g] [%] [mOsm / kg]

SPC / CH / SPG * 239 ± 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 / DSPG * 255 ± 11 96.4 ± 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 / SPG * 209 100.8 37.9 -

6: 3: 1

EPC / CH / SPG * 347 90, 9 60.9 387 6: 3: 1 amount of iodine / lipid used = 1: 1.5 amount of iodine / lipid used = 1: 1 extrusion at room temperature Example 2 Storage stability of lopromide-containing liposome suspensions

Liposomes prepared according to Example 1 (SPC / CH / SPG 6: 3: 1, amount of iodine / lipid used 1: 1.5) are stored in the refrigerator or at room temperature and their stability is examined after 9 months.

Example 3: Preparation of Gd-containing liposome suspensions

Liposomes with Gd-containing MRI contrast media are produced by means of the high-pressure extrusion process described in Example 1.

Lipid contrast ¬ 0 Gd content Ver Osmo- compound encapsulation lality settlement * [nm] [μmol / ml]

[%] [mOsm / 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 / SPG Gado-227 80.2 41.0 not

6: 3: 1 butrol determined

SPC / CH / SPG 141 80.6 49.5 138

6: 3: 1

SPC / CH / SPG # 210 ± 7 110.8 ± 2.3 57.4 ± 1.5 206 ± 5

7: 2: 1 amount of iodine / lipid used = 1: 1.5 n = 3, all others n = 1

Example: Plasma stability of lopromide-containing liposomes

Liposome suspensions prepared according to Example 1 (batch A: 198 nm, 42.9% encapsulated, batch B: 103 n, 34.7% encapsulated) were mixed with human plasma, an iodine concentration of about 5 mg / ml being set. Each 1 ml of this plasma contrast medium mixture was then dialyzed in a Dianorm equilibrium dialysis apparatus (Dianorm, Heidelberg) against the corresponding human plasma through dialysis membranes with a cutoff of 5000 Da (Dianorm). At different times, samples were taken from the retentate and permeate side and the iodine content was determined by means of X-ray fluorescence spectroscopy (FEA). The results obtained can be seen in FIG. 1.

Example 5 Organ Distribution of Lopromide-Containing Liposomes in the Rat

The liposome suspension (approach A) listed in Example 4 was injected at a dose of 250 mg total iodine / kg into 16 male rats (weight: 137-160 g) and 4 animals each 0.25; 1; Killed 4 and 24 hours after the injection. The liver, spleen, lungs and blood were then examined for their iodine content by means of FEA. The results (% of the dose / organ administered) are shown in the table below.

Time 0.25 1 4 24 p.i. [H]

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

Lungs 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 3.5 ± 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% DSPE-PEG 2000 - 204 nm, 45% encapsulated) was administered in a dose of 250 mg total iodine / kg to 16 male rats (weight: 136-160 g) and 4 animals 0.25 each; 1; Killed 4 and 24 hours after the injection. The liver, spleen, lungs and blood were then examined for their iodine content by means of FEA. The results (% of the dose / organ administered) are shown in the table below.

Example 7: Blood-pool imaging (CT) in rabbits

The liposome suspension (approach A) listed in Example 4 was examined in a dose of 200 mg total iodine / kg in the rabbit. The monomeric X-ray contrast agent Ultravist® (Iopro id (INN)) was used as a control. The X-ray density in Hounsfield Units (HU) in the aorta and in the liver tissue was measured from 0 to 20 minutes after a single intravenous application (spiral CT, Somatom plus S, Siemens, at 120 kV). The area under the

Signal-time curve for aorta and for liver over 20 minutes calculated in HU * min; the difference between aorta and liver tissue is a measure of the contrast quality. The results were shown in Figure 2. As is clear from FIG. 2, the signal difference between the aorta and the liver is significantly higher for the iopromide liposomes than for the free iopromide (Ultravist®).

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 Example 3) in human plasma was investigated as described in Example 4. The Gd concentration used at the GGD was 20 μmol Gd / ml. The result was shown in Figure 3.

Example 9: Blood level profiles after administration of Gd-EOB-DTPA-containing liposomes to the rat

The liposome suspensions listed in Example 8 (batches A and B) were injected at a dose of 0.3 mmol total Gd / kg into 16 male rats (weight: 137-158 g) and 4 animals each 0.25; 1; Killed 4 and 24 hours after the injection. The Gd content in the blood was then determined by means of ICP-AES (inductively coupled plasma atomic emission spectroscopy). The liver concentrations were not shown here, since the unencapsulated Gd-EOB-DTPA also accumulates specifically in the liver (liver contrast agent for MRI). The results (% of the applied dose / 31ut) are shown in the table below.

Time p.i. [] 0.25 1 4 24

(MW ± SD) (MW ± SD) (MW ± SD) (MW ± SD)

Approach A 35.9 ± 0.6 21.5 ± 1.2 10.2 ± 2.3 2.1 ± 0.5

Approach 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

Rabbit liposomes

A Gd-EOB-DTPA-containing liposome suspension (approach A, see Example 8) was administered in a dose of 0.3 mmol Gd / kg i.v. (Anterior vein) administered to an anesthetized rabbit (3 ml / min). Since Gd-EOB-DTPA, which is actually used as an MRI liver contrast medium, also absorbs X-rays, it was possible to use computer tomography (CT) to test the accumulation of the liposomal component in the blood. FIG. 4 shows the course of the X-ray density in ΔHU in the aorta over a period of one hour.

Claims

claims

1. active ingredient-containing liposome formulation, characterized in that a) the following mixing ratio of the 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 substance is an X-ray or MRI contrast medium or a radio diagnostic agent.

2. active ingredient-containing liposome formulation, characterized in that a) the following mixing 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 substance is an X-ray or MRI contrast medium or a radio diagnostic agent.

3. active ingredient-containing liposome formulation, characterized in that a) the following mixing ratio of the lipids is present: 60-70% phosphatidylcholine,

20-30% cholesterol,

4. active ingredient-containing liposome formulation according to any one of claims 1-3, characterized in that phosphatidylcholine, phosphatidylethanolamine, soybean phosphatidylcholine, Egg phosphatidycholine or em sphmgolipid is used as phospholipid.

5. Active ingredient-containing liposome formulation according to any one of claims 1-3, characterized in that hexadecyl poly (3) glycerol, dialkylpol (7) glycerol ether or an alkyl glucoside is used as the amphiphilic substance.

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

7. active ingredient-containing liposome formulation according to any one of claims 1-3, characterized in that a fatty acid, dicetyl phosphate, cholesterol isocyanate, phosphatidylglycerol, phosphatidylserm, phosphatidic acid or phosphatidylmositol is used as charge carrier.

8. active ingredient-containing liposome formulation according to any one of claims 1-3, characterized in that at least one amphiphilic substance is used as charge carrier.

9. Active ingredient-containing liposome formulation according to one of the

Claims 1-3, characterized in that the liposomes have an average diameter of 150-250 nm.

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

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

12. Active ingredient-containing liposome formulation according to any one of claims 1-3, characterized in that 40-65% of

Active ingredient are encapsulated in the liposomes.

13. Active ingredient-containing liposome formulation according to claim 1, characterized in that the following mixing ratios of the components are present: a) 60% soy phosphatidycholine, 30% cholesterol, 10% soy phosphatidylglycerol, b) 70% soy phosphatidycholine, 20% cholesterol, 10% soy phosphatidyl glycerol, c) 75% Soy phosphatidycholine, 20% cholesterol,

5% soy phosphatidylglycerol, d) 50% soy phosphatidycholine, 40% cholesterol, 10% soy phosphatidylglycerol, e) 60% soy phosphatidycholine, 30% cholesterol, 10% distearoylphosphatidylglycerol, f) 70% soy phosphatidroglyphyl, 20% chololol, 20% chololol, 20% Soybean Phosphatidycholine, 30% Cholesterol, 10% Di yristoylphosphatidylglycerol, h) 60% Soybean Phosphatidycholine, 30% Cholesterol, 10% Distearoylphosphatidic Acid, i) 70% Soybean Phosphatidycholine, 20% Cholesterol, 10% Distearoylphosphatidoloyl, 5% Soyaphosphateidol, 5% % Distearoylphosphatidic acid.

14. Active ingredient-containing liposome formulation according to any one of claims 1-3, characterized in that the active ingredient

Amidotrizoate, metrizoate, iopromide, iohexol, iopamidol, iosimide, ioversol, iomeprol, iopentol, ioxilan, iobitridol, ioxaglate, iotrolan, iodixanol, bis- [{3-N- (2, 3-dihydroxy-propyl-carbamoyl carbamoyl} -2, 4, 6-triiod-N- (2, 3-di-hydroxypropyl) -anilide] - alonic acid or 5-hydroxyacetyl-amino-2, 4, 6-triiodo-isophthalic acid- [(2, 3- dihydroxy-N-methylpropyl) - (2, 3-dihydroxypropyi)] diamide.

15. Active ingredient-containing liposome formulation according to any one of claims 1-3, characterized in that the active ingredient Gd-DTPA, Gd-EOE-DTPA, Yb-EOB-DTPA, Dy-EOB-DTPA, Gd-DOTA, Gd-BOPTA, Gadobutrol or Mn-DPDP is.

16. Use of active ingredient-containing liposome formulation for the preparation of an agent for X-ray diagnosis, characterized in that a) the following mixing ratio of the 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 substance is an X-ray contrast agent.

17. Use of active ingredient-containing liposome formulation for the production of an agent for X-ray diagnosis, characterized in that a) the following mixing ratio of the lipids is present: 40-70% phospholipids or ampniphiles,

30-50% sterols, 5-20% charge carriers, b) the liposomes have an average diameter of 100-400 nm and c) the active substance is an X-ray contrast agent.

18. Use of active ingredient-containing liposome formulation for the preparation of an agent for X-ray diagnosis, characterized in that a) the following mixing ratio of the lipids is present: 60-70% phosphatidylcholine,

20-30% cholesterol,

2-10% phosphatidylglycerol, phosphatidic acid and / or cholesterol isuccinate, b) the liposomes have an average diameter of 150-350 nm and c) the active substance is an X-ray contrast agent.

19. Use of active ingredient-containing liposome formulation according to one of claims 16-18 for the production of an agent for the X-ray diagnosis of the intravascular space.

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

21. Use of active ingredient-containing liposome formulation according to claim 20 for the production of an agent for the X-ray diagnostic imaging of the intravascular space by means of computer tomography.

22. Use of active ingredient-containing liposome formulation for: the preparation of an agent for MRI diagnostics, characterized in that a) the following mixing ratio of the 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 substance is an MRI contrast agent.

23. Use of active ingredient-containing liposome formulation for. the preparation of an agent for MRI diagnostics, characterized in that a) the following mixing 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 Have 100-400 nm and c) the active substance is an MRI contrast medium.

24. Use of active ingredient-containing liposome formulation for the production of an agent for MRI diagnostics, characterized in that a) the following mixing ratio of the lipids is present: 60-70% phosphatidylcholm,

20-30% cholesterol,

2-10% phosphatidylglycerol, phosphatidic acid and / or cholesterol hemisuccin, b) the liposomes have an average diameter of 150-350 nm and c) the active substance is an MRI contrast agent.

25. Use of active ingredient-containing liposome formulation according to one of claims 22-24 for the preparation of an agent for the diagnostic imaging of the intravascular space by means of MRI.

26. Use of active ingredient-containing liposome formulation for the production of an agent for radio diagnostics, characterized in that a) the following mixing ratio of the 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 substance is a radio diagnostic agent.

27. Use of active ingredient-containing liposome formulation for the production of an MpEEel for radio diagnostics, characterized in that a) the following mixing 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 radio diagnostic agent.

28. Use of active ingredient-containing liposome formulation for the production of an agent for radio diagnostics, characterized in that a) the following mixing ratio of the lipids is present: 60-70% phosphatidylcholine,

20-30% cholesterol,

2-10% phosphatidylglycerol, phosphatidic acid and / or cholesterol isuccinate, b) the liposomes have an average diameter of 150-350 nm and c) the active ingredient is a radio diagnostic agent.

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