WO1998023297A2

WO1998023297A2 - Particulate compositions

Info

Publication number: WO1998023297A2
Application number: PCT/GB1997/003283
Authority: WO
Inventors: Liv Ingrid ØDEGÅRDSTUEN; Kari Dyvik; Kathrin Bjerknes; Anne Kjersti Fahlvik
Original assignee: Nycomed Imaging AS
Current assignee: GE Healthcare AS
Priority date: 1996-11-29
Filing date: 1997-11-28
Publication date: 1998-06-04
Anticipated expiration: 1999-05-29
Also published as: AU5129398A; GB9624918D0; JP2001504837A; EP1009445A2; WO1998023297A3

Abstract

The invention provides a contrast agent composition, preferably an X-ray contrast medium, comprising vesicles in suspension in an aqueous medium, said vesicles and optionally also said aqueous medium containing an iodinated X-ray contrast agent, characterised in that a dose of said composition containing 100 mgI entrapped in said vesicles contains no more than 8x1012 vesicles.

Description

Particulate Compositions

This invention relates to parenterally administrable particulate compositions, in particular contrast media, eg. MRI, X-ray, ultrasound, light imaging or nuclear imaging contrast media, especially X-ray contrast media and more particularly vesicle containing X-ray contrast media .

Contrast media are widely used in a range of imaging modalities (eg. CT, MRI, ultrasound etc.) in order to improve the contrast in the images obtained, for example to assist in differentiation between different organs or between healthy and unhealthy tissue.

In the field of X-ray imaging, the commercial contrast agents fall into two major categories, insoluble inorganic heavy metal salts (eg. barium sulphate) for imaging of the gastrointestinal (GI) tract and soluble iodinated organic compounds (usually triiodophenyl monomers and dimers, such as iohexol and iodixanol) for parenteral administration or also for GI imaging.

In view of their hydrophilic small molecular nature, the soluble iodinated organic compounds have similar biodistribution and bioelimination patterns following parenteral administration, distributing into the extracellular fluid (ECF) compartment before relatively rapidly being renally excreted. Accordingly their clinical indications are similar although the non-ionic compounds (such as iohexol) are greatly preferred over the ionic compounds (such as metrizoate) for parenteral administration due to better patient comfort and tolerance. There is however an increasing demand for organ specificity for new contrast media and in particular there are demands for targetting agents which enhance contrast of specific organs such as the liver (liver agents) , the vasculature (blood pool agents) and the lymph nodes (lymphatic agents) .

To meet the liver demands, various attempts have been made to formulate particulate X-ray contrast agents which, due to their particulate nature, are abstracted from the blood by the reticuloendothelial system and hence accumulate at the liver. To meet the demand for a blood pool agent, iodophenyl contrast agents have been associated with macromolecular substrates or structures to extend their vascular half-lives. For lymph node imaging, attempts have been made to use local administration (eg. subcutaneous administration) of particulate contrast media. However, lymph node imaging following intravascular administration of particulate agents is also feasible.

Potential liver agents based on biodegradable particles (see WO 89/00988 and WO 90/07491) , on liposomes containing X-ray contrast agents (see WO 95/26205) and on emulsions (see EP-A-294534) have been proposed but, as yet, no such agents have reached the market. There thus remains an unfulfilled need for efficient and stable non-toxic organ-specific X-ray contrast agents.

Vesicle containing agents, eg. liposomal agents, such as described in WO 95/26205 have shown promising liver imaging results in animal studies and acceptable safety profiles in pre-clinical trials. In trials on human volunteers at diagnostically desirable dosages, however, minor but uncomfortable adverse events have occurred.

Thus at dosages of 70 to 100 mgl/per kg bodyweight a liposomal contrast agent containing 80 mg I/mL of a water-soluble non-ionic X-ray contrast agent, entrapped within phospholipid liposomes (having a mean (Z-average) particle size of about 350 nm, and constituting about 40% of the total composition volume) and dissolved in the aqueous suspension medium for the liposomes at substantially equal concentration caused side effects such as fever, chills, headache, nausea and vomiting. Investigations now indicate that these effects are related to the total particle count administered to the patient .

It is an objective of the present invention to provide a particulate contrast medium which at a diagnostically effective iodine dosage, does not provoke such side effects, or for which the side effects are reduced to a tolerable level.

This objective is met by the present invention by the reduction of the particle count/mg enclosed iodine in the composition, eg. by the use of higher entrapped iodine concentrations and/or by the use of higher volume vesicles .

Thus viewed from one aspect the invention provides a contrast agent composition, preferably an X-ray contrast medium, comprising vesicles (eg. micelles or liposomes) in suspension in an aqueous medium, said vesicles and optionally also said aqueous medium containing an iodinated X-ray contrast agent, characterised in that a dose of said composition containing 100 mgl entrapped in said vesicles contains no more than 8xl0¹² vesicles (preferably less than 6xl0¹², especially preferably less than 4xl0¹², particularly preferably less than 2xl0¹², and more especially less than 10¹², eg. 3 to 9xl0 , more preferably lxlO⁷ to 9X10¹¹, eg. lxlO⁷ to 9.8xl0⁹ vesicles) . Preferably the weight ratio of encapsulated iodine to vesicle membrane material (eg. lipid) is at least 1.8:1, eg. at least 2:1, for example 2-5:1, more preferably 2.5 to 4.8:1, especially preferably 3.0 to 4.5:1. Preferably, in the compositions at least 40% of the iodine content is in the vesicles . Preferably the encapsulated iodine content is at least 30 mg/mL (of total composition) , more preferably at least 60 mg/mL and particularly preferably at least 100 mg/mL.

Viewed from a further aspect the invention also provides a method of generating a contrast enhanced image, eg. an X-ray image, comprising parenterally (eg. intravenously or intraarterially) administering a contrast agent composition according to the invention to a human or non-human animal (preferably mammal) body and generating an image, preferably an X-ray (eg. CT) image, of at least part of said body, preferably at least of the liver.

In the compositions of the invention, the vesicles may be unilamellar or multilamellar; preferably the composition has a high degree of bi or trilamellar liposomes and/or unilamellar vesicles. In the vesicles (the term vesicle is used herein to designate both unilamellar and multilamellar structures) the iodine may be entirely or substantially entirely associated with the vesicles, eg. by encapsulation within the vesicle core and/or by incorporation within the vesicle membrane .

Particularly preferably however the aqueous medium within which the vesicles are suspended will contain a dissolved iodinated organic contrast agent, especially at or near the iodine concentration within the vesicle core. In this way, the contrast medium has a three-fold contrast effect - the vesicles providing blood pool and RES (eg. liver and lymph node) contrast enhancement and the iodinated agent in the aqueous suspension medium providing blood pool and other ECF contrast . The iodine concentration in the composition as a whole is conveniently 100 to 500 mgl/mL, especially 100 to 460 mgl/mL, more especially 150 to 420 mgl/mL, particularly 200 to 400 mgl/mL, and more particularly 225 to 370 mgl/mL. The concentration of vesicle encapsulated contrast medium is preferably at least 40 mgl/mL (eg. 40 to 160 mgl/mL) , especially preferably at least 40 to 100 mgl/mL relative to the volume of the overall composition. When administered for delayed or dynamic liver imaging, the dosage is preferably 60-600 mgl (in total) per kg bodyweight, preferably 150 to 500 mgl/kg, more preferably 200 to 400 mgl/kg. At these higher iodine concentrations, injection or infusion, and in particular bolus injection of the contrast medium provides particularly diagnostically effective iodine concentrations within the vasculature.

Having substantially similar iodine concentrations within and surrounding the vesicles has further advantages in terms of simplicity of production and sterilization, particularly autoclave sterilization, of the contrast medium since removal of non-entrapped iodine is not required and since diffusion over time (and hence storage) will not radically change the iodine distribution.

The particle count, eg. number of vesicles per mL, in the contrast medium of the invention is not in itself a critical parameter. What is critical, and therefore characteristic of the compositions of the invention, is the number of particles per dose (or more particularly in the dose per kg bodyweight of the patient) .

The present inventors surmise that the adverse effects and the lower imaging effects of the prior art parenteral particulate X-ray contrast media arise from overloading of the particle uptake system of the liver and that therefore what is critical is to deliver to the liver an iodine dose which is diagnostically effective for liver contrast enhancement without causing a level of uptake saturation sufficient to provoke the unacceptable side effects.

The X-ray contrast agent used in the compositions of the invention is preferably a triiodophenyl compound and more especially a non-ionic compound. Both monomers (such as iohexol) and dimers (such as iodixanol) may be used. Examples of suitable X-ray contrast agents include the compounds of WO-A-96/09282 and WO-A-96/09285 as well as compounds such as iodixanol, iopentol, iohexol, metrizamide, metrizoate, ioversol, ioxilan, ioxaglate, iopamidol, iotrolan, ioglicate, iomeprol, iophendylate, iopromide, iosimide, iotasul, iothalamate, iotroxate and ioxitalamate . Non-ionic monomers such as iohexol, iopamidol and iopentol and non-ionic dimers such as iodixanol and iotrolan are however most preferred.

The vesicles, used in the compositions of the invention as the vehicles for transporting encapsulated iodine to the liver, may be produced by conventional means using vesicle membrane forming agents, preferably phospholipids . Examples of suitable membrane forming materials are set out in WO 95/26205 the disclosure of which is incorporated herein by reference. Thus the membrane preferably comprises both a neutral phospholipid and a charged phospholipid. Moreover it is preferred that both phospholipids are substantially saturated.

The neutral phospholipid preferably comprises at least one substantially saturated fatty acid residue. The number of carbon atoms in such fatty acid residues is preferably at least 15 or more, preferably at least 16. Where the number of carbon atoms in a fatty acid residue is less than 14, the ability of liposomes to hold the internal aqueous phase is low and the stability of liposomes in blood after administration is low. On the other hand, where the number of carbon atoms in a fatty acid residue is 28 or more, biocompatibility becomes low, and very high temperature is necessary during production of liposomes.

The term "substantially saturated" as used above means that fatty acid residues of the neutral and charged phospholipids are fully saturated (i.e. contain no C-C double bonds) or that the extent of their unsaturation is very low, e.g. as shown by an iodine value of no more than 20, preferably no more than 10 most preferably 5 or less. Where the extent of unsaturation is too high, the liposomes are easily oxidized.

The vesicle membrane advantageously comprises a charged phospholipid, eg. one comprising at least one substantially saturated fatty acid residue. The number of carbon atoms in such a fatty acid residue is usually at least 14, preferably at least 15 and more preferably at least 16, for the reasons set out above.

The absolute value of the zeta potential of the vesicles in the compositions of the invention is preferably less than -50 mV, particularly less than -58 and especially less than -60, eg. -50 to -70 mV.

Neutral phospholipids useful in the present invention include, for example, neutral glycerophospholipids, for example a partially or fully hydrogenated naturally occurring (e.g. soybean- or egg yolk-derived) or synthetic phosphatidylcholine, semi-synthetic or synthetic dipalmitoyl phosphatidylcholine (DPPC) or distearoyl phosphatidylcholine (DSPC) .

Charged phospholipids useful in the present invention include, for example, positively or negatively charged glycerophospholipidε . Negatively charged phospholipids include, for example, phosphatidylserine, for example a partially or fully hydrogenated naturally occurring (e.g. soybean- or egg yolk-derived) or semi-synthetic phosphatidylserine, particularly semi-synthetic or synthetic dipalmitoyl phosphatidylserine (DPPS) or distearoyl phosphatidylserine (DSPS) ; phosphatidylglycerol, for example a partially or fully hydrogenated naturally occurring (e.g. soybean- or egg yolk-derived) or semi-synthetic phosphatidylglycerol, particularly semi-synthetic or synthetic dipalmitoyl phosphatidylglycerol (DPPG) or distearoyl phosphatidylglycerol (DSPG) ; phosphatidylinositol, for example a partially or fully hydrogenated naturally occurring (e.g. soybean- or egg yolk-derived) or semi- synthetic phosphatidylinositol, particularly semi- synthetic or synthetic dipalmitoyl phosphatidylinositol (DPPI) or distearoyl phosphatidylinositol (DSPI) ; phosphatidic acid, for example a partially or fully hydrogenated naturally occurring (e.g. soybean- or egg yolk-derived) or semi-synthetic phosphatidic acid, particularly semi-synthetic or synthetic dipalmitoyl phosphatidic acid (DPPA) or distearoyl phosphatidic acid (OSPA) . Although such a charged phospholipid is commonly used alone, more than one charged phospholipid may be used. In the case where more than one charged phospholipid is used, preferably both the charged phospholipids are positively charged, or both the charged phospholipids are negatively charged, in order to prevent aggregation. Positively charged lipids include, for example, an ester of phosphatidic acid with an aminoalcohol, such as an ester of dipalmitoyl phosphatidic acid or distearoyl phosphatidic acid with hydroxyethylenediamine .

According to the present invention, the ratio of the neutral phospholipid to the charged phospholipid is usually 200:1 to 3:1, preferably 60:1 to 4:1, and more preferably 40:1 to 5:1 by weight, e.g. about 10:1.

The mean vesicle size is preferably in the range 50 to 4000 nm, especially 100 to 3000 nm particularly 150 to 2000 nm. The upper size limit is dictated partially by the size constraints of the capillaries and may be exceedable for vesicles with particularly flexible membranes. The lower size limits are dictated by the desire according to the invention to minimize the number of vesicles required to deliver a diagnostically effective iodine dosage to the liver and other parts of the RES. Size discrimination of vesicles may be achieved by extrusion of larger vesicles through filters of appropriate pore sizes (see Biochem Biophys Acta 557:9 (1979)), or by gravitational or centrifugal separation techniques .

The contrast agent compositions of the invention may contain various optional components in addition to the membrane forming agents and the X-ray contrast agent. For example, vitamin E (α-tocopherol) and/or vitamin E acetate ester as an antioxidant may be added in an amount of 0.01 to 2 molar %, preferably 0.1 to 1 molar % relative to total amount of lipids . Similarly sorbitol is preferably included as a tonicity adjusting agent (particularly important where a non-ionic dimer is used as the X-ray contrast agent) . For diagnostic compositions comprising vesicles containing the above-mentioned phospholipids, the concentration of total lipid is generally 10 mg/ml to 100 mg/ml, preferably 15 mg/ml to 90 mg/ml, and more preferably 20 mg/ml to 80 mg/ml, in order to enhance encapsulation of contrast agent in the vesicles.

Such contrast agents are preferably encapsulated in the vesicles in the form of an isotonic solution or suspension (relative to physiological osmotic pressure in the body) in an appropriate medium so that the vesicles are stably maintained in the body after administration. As a medium, water, buffer solution such as TRIS-HC1 buffer, phosphate buffer, citrate buffer or the like may be used.

A preferred pH range at room temperature is 4.5-8.5, more preferably 6.8-7.8. Where the contrast agent is a non-ionic X-ray contrast agent carrying multiple hydroxyl groups, e.g. iohexol, iodixanol or iopamidol, the buffer is preferably one having a negative temperature coefficient, as described in US-A-4278654. Amine buffers have the required properties, particularly TRIS. This type of buffer has a lower pH at autoclaving temperatures, which increases the stability of the X-ray contrast agent during autoclaving, while returning to a physiologically more acceptable pH at room temperature.

To obtain an isotonic solution or dispersion, the contrast agent is dissolved or suspended in a medium at a concentration which provides an isotonic solution. In the case where a contrast agent alone cannot provide an isotonic solution because, for example, solubility of the contrast agent is low, other non-toxic water soluble substances, for example salts such as sodium chloride or sugars such as mannitol, glucose, sucrose, sorbitol or the like may be added to the medium so that an isotonic solution is formed.

One particular advantage of the compositions of the invention which are also in accordance with WO 95/26205 is their ability to withstand autoclaving. They also have a high encapsulation capacity and encapsulation ratio by virtue of their lipid composition.

The vesicles can be produced by conventional procedures used for formation of multilamellar liposomes. These procedures include the Bangham method (J. Mol . Dial. 13 , 238-252, 1965), the polyvalent alcohol method (Japanese Examined Patent Publication (Kokoku) No. 4-36734) , the lipid-solution method (Japanese Examined Patent Publication (Kokoku) No. 4-36735), and the mechanochemical method (Japanese Examined Patent Publication (Kokoku) No. 4-28412) .

Generally, desired multilamellar vesicles can be prepared by dissolving the above-mentioned phospholipids in a volatile organic solvent such as chloroform, methanol, dichloromethane, ethanol or the like, or a mixed solvent of said organic solvent and water, removing said solvent, mixing the resulting residue with an aqueous phase containing a contrast agent, and shaking or stirring the mixture.

As the step for removing solvent in the above-mentioned process, Bangham¹ s method uses evaporation, but spray- drying or lyophilization also can be used.

In the above-mentioned vesicle-preparing processes, the amount of the solvent used relative to lipid is not critical, and any amount which allows dissolution of lipid is acceptable. Removing solvent from the resulting mixture of lipid and solvent by evaporation can be carried out according to conventional procedure,

Claims

such as evaporation under reduced pressure or, if necessary in the presence of inert gas. In practice, the above-mentioned volatile organic solvents may be used, if desired in mixed solvents comprising 10 volumes of said organic solvent and up to 1 volume of water.To effect solvent removal by lyophilization, a solvent is selected which can be removed at a reduced pressure of about 0.005 to 0.1 Torr at a temperature lower than the freezing point of the solvent, typically -30°C to -50°C. If necessary a cryoprotectant, eg. trehalose or sucrose, may be added. Where solvent removal is effected by spray drying the air pressure is typically controlled to 1.0 kg/cm2 and the air flow rate to 0.35 cm2/minute, the inlet temperature being adjusted to a temperature higher than the boiling point of the solvent used. For example, the solvent may be chloroform, the temperature may be adjusted to 60 to 90°C, and the spray drying may be effected according to conventional procedures .The lipid residue thus obtained is mixed with an aqueous solution containing a contrast agent at a temperature equal to or higher than the phase transition temperature (Tc) of the lipid used, and then the mixture is vigorously or more gently shaken or stirred at a temperature equal to or higher than said Tc to produce the desired vesicles suspended in the aqueous solution containing the contrast agent . The electrolyte ion concentration in the aqueous solution containing the contrast agent should desirably be as low as possible to avoid adversely affecting the encapsulation efficiency etc.; generally the total concentration of positive and negative ions apart from the contrast agent is desirably not more than about 40 mM, preferably being not more than about 20 mM. The vesicles thus prepared are present as a suspension in an aqueous medium (outer liquid) , and are generally used as such as a diagnostic composition. The solution of the contrast agent which has not been encapsulated in vesicles during the formation of the vesicles is present as the outer liquid. Alternatively, the outer liquid may be replaced with another liquid, although the concentration of imaging agent should desirably be the same in the outer liquid as in any inner liquid. In any case, the outer liquid (dispersion medium) is preferably isoosmotic relative to the internal liquid phase of the vesicles. The electrolyte ion concentration in the thus-prepared suspension should desirably be as low as possible; generally the total concentration of positive and negative ions apart from the contrast agent is desirably not more than about 40 mM, preferably being not more than about 20 mM, in order to enhance the stability of the vesicles on heat sterilisation and long term storage.The encapsulation capacity of the vesicles is generally at least 5 ml/g lipid, preferably at least 6 ml/g. When encapsulating an iodinated X-ray contrast agent by the method of the invention, the concentration of the contrast agent in the stock aqueous solution should desirably be high eg. 200 to 460 mgl/mL, preferably 250 to 400 mgl/mL.The most preferred X-ray contrast compositions of the invention comprise liposomes having a neutral phospholipid (eg. hydrogenated phosphatidyl choline) and a charged phospholipid (eg. hydrogenated phosphatidylserine) in a weight ratio of about 10:1. Such a composition may have an encapsulation capacity over 7 ml/g. The most preferred X-ray contrast agents for use in such compositions are iodixanol and iohexol. The aqueous medium inside and outside the liposomes preferably contains about 300-400 mgl iodixanol/ml, as well as an isotonicity adjusting agent such as sorbitol, a stabilising agent such as EDTANa2Ca and TRIS buffer (pH about 7.4).While the invention has been described in terms of vesicle compositions, it also extends to other particulate containing aqueous compositions for parenteral administration (especially intravenous and intraarterial administration) , where the particles are or contain an X-ray contrast agent, eg. an iodinated compound. Such particles may be the droplets of an emulsion, eg. of a water-insoluble liquid X-ray contrast agent, or they may be solid particles of a water- insoluble solid X-ray contrast agent. Such particles should have an iodine content such that a 100 mgl dose is contained in not more than 8xl012 particles (preferably less than 6xl012, especially preferably less than 4xl012 and particularly preferably less than 2xl012, more especially less than 1012 eg. 0.3 to 4.5xl012 vesicles) . Again such compositions will desirably also contain an iodinated X-ray contrast agent dissolved in the aqueous phase, eg. providing up to 60% of the total iodine dose .The documents referred to herein are incorporated herein by reference.The invention will now be described further with reference to the following Examples and the accompanying drawings in which:Figure 1 shows a plot of liver: spleen distibution ratio against particle size (Dz) for formulations according to the invention four hours after intravenous administration to the rat. The suspension of Example 1 is the same as that of Examples 9 and 12. The suspension of Example 2 is the same as that of Examples 13 and 17. The suspension of Example 5 is the same as that of Examples 10 and 14. The suspension of Example 11 is the same as that of Examples 15 and 19.EXAMPLES 1 (COMPARATIVE) TO 4Iodixanol solutions were prepared in a beaker during stirring and heating up to 95°C, until transparent solutions were obtained. The most concentrated iodixanol solution was dissolved in excess water with the excess being evaporated afterwards. The solutions were stored overnight in the dark at 4°C. Sorbitol was then added if necessary in order to adjust tonicity. The iodixanol solutions were added to a glassy reactor with a heating jacket connected to a water bath at 80°C. The phospholipid mixture (hydrogenated egg phosphatidylcholine/hydrogenated egg phosphatidylserine sodium in an 11:1 weight ratio) was added to the solutions during stirring. The dispersions were heated to 65-76°C during stirring before further stirring for at least 20 minutes. The dispersions were homogenized using a high-speed rotor stator (Ystral) at 13000 rpm, before being transferred to a pressure tank with a heating jacket at 80°C. The dispersions were extruded at 3-6 Bar through seven 1.0 μm polycarbonate filters. After cooling the liposomal suspensions to below 50°C, the TRIS/EDTA solution was added and the preparations were autoclaved.The compositional details are set out in Tables 1 and 2 below. Table 1Table 2* Estimated number of particles for 100 mg entrapped iodine dose EXAMPLE 2Liposomes are made as described in Example 1 but extruded through larger diameter pores, 3 to 5 μm rather than 1 μm. Particles in the lower size range are then removed by centrifugation or cross flow filtration to yield particles with an encapsulated iodine to lipid ratio of 3.6, mean volume diameter 0.95 μm.For such particles the number required for a 100 mg encapsulated iodine dose is 9.2xl09.EXAMPLES 5 TO 8Aqueous iohexol solutions were prepared in a beaker with stirring and heating to 95°C until a transparent solution was obtained. Sorbitol was then added if necessary to adjust the tonicity.The iohexol solution was added to a glass reactor with a heating jacket connected to a water bath at 80°C. A lipid mixture (as in Examples 1 to 4) was added to the solution during stirring. The dispersion was heated to 65-75°C during stirring before further stirring for at least 20 minutes. The dispersion was homogenized using a high-speed rotor stator (Ystral) at 13000 rpm before being transferred to a pressure tank with a heating jacket at 80°C.The dispersion was extruded at 3-6 Bar through 7 polycarbonate 1.0 μm filters. After cooling the liposomal suspension to below 50°C, the TRIS/EDTA solution was added before filling into vials. The vials were continuously shaken during autoclaving.The dispersion of Examples 5 and 8 were administered by intravenous injection to rats at a dosage of 100 mg encapsulated I/kg bodyweight. Table 3Table 4 n.p . determnat on not performedEXAMPLES 9 (COMPARATIVE) -11After intravenous injection to the rabbit of the liposomes listed below at a dose of 100 mg encapsulated I/kg bodyweight, there was specific accumulation of the liposomes in normal liver tissue in rabbits leading to a contrast enhancement in excess of 40 HU (Houndsfield units) . As metaεtatic lesions within the liver do not take up liposomes, there would be an enhanced visualization of the lesions following injection of the liposomes.Imaging was performed on a Philips Tomoscan SR7000. 5 mm slice thickness, 1 second scan-time, four rabbits per group . Table 5Table 6Δ = change from base lineEXAMPLES 12 (COMPARATIVE) TO 15 AND 16 (COMPARATIVE) TO 20After an intravenous administration to the rat of 100 mg encapsulated I per kg bodyweight, the encapsulated iodixanol is mainly taken up by Kupffer cells in the liver and filtered by the spleen with subsequent Kupffer cell uptake. The specific uptake of liposomes in normal liver have been demonstrated both in rats and rabbits to give rise to a contrast enhancement in excess of 40 HU. The iodine concentration giving rise to this contrast enhancement is at least 1 mg I/g liver. As demonstrated in Tables 7 to 10, the formulations used resulted in an iodine concentration in the liver of at least 1 mg I/g tissue. The correlation between particle size distribution and liver/spleen ratio four hours after admininstration is shown in Figure 1 of the accompanying drawings .Table 7Table 8Maximal concentration (mg/g) and total amount (% of dose) of iodine in the rat liver at 4 hours after a single i.v. administration of 100 mg encapsulated I per kg bodyweight . Table 9Table 10Iodine to lipid ratio and amount of lipid (mg/kg bodyweight) at a dose of 100 mg encapsulated I per kg bodyweight .Cmax = mean maximal concentration observed in the rat liver (using 3 rats per study) . Claims :

1. A contrast agent composition comprising vesicles in suspension in an aqueous medium, said vesicles and optionally also said aqueous medium containing an iodinated X-ray contrast agent, characterised in that a dose of said composition containing 100 mgl entrapped in said vesicles contains no more than 8xl0¹² vesicles.

2. A composition as claimed in claim 1 containing less than 10¹² vesicles in a dose containing lOOmg I entrapped in said vesicles.

3. A composition as claimed in claim 1 containing 10⁷ to 9.8xl0⁸ vesicles in a dose containing 100 mg I entrapped in said vesicles.

4. A composition as claimed in any one of claims 1 to 3 having a weight ratio of vesicle encapsulated iodine to vesicle membrane material of at least 1.8:1.

5. A composition as claimed in any one of claims 1 to 3 having a weight ratio of vesicle encapsulated iodine to vesicle membrane material of at least 2:1.

6. A composition as claimed in any one of claims 1 to

5 having a vesicle encapsulated iodine content of at least 30 mg/mL.

7. A composition as claimed in any one of claims 1 to

6 having an iodine content of 100 to 500 mg/mL.

8. A composition as claimed in any one of claims 1 to

7 wherein said iodinated X-ray contrast agent is a non- ionic, triodophenyl group containing compound.

9. A composition as claimed in any one of claims 1 to 7 wherein said iodinated X-ray contrast agent is selected from iodixanol, iopentol, iohexol, metrizamide, metrizoate, ioversol, ioxilan, ioxaglate, iopamidol, iotrolan, ioglicate, iomeprol, iophendylate, iopromide, iosimide, iotasul, iothalamate, iotroxate and ioxitalamate .

10. A compound as claimed in any one. of claims 1 to 9 wherein the membrane of said vesicles comprise a neutral and a charged phospholipid.

11. A composition as claimed in any one of claims 1 to

10 wherein said vesicles have a mean size of 150 to 2000 nm.

12. A composition as claimed in any one of claims 1 to

11 being an X-ray contrast medium.

13. A method of generating a contrast enhanced image comprising parenterally administering a contrast agent composition as claimed in any one of claims 1 to 12 to a human or non-human animal body and generating an image of at least part of said body.

14. A method as claimed in claim 13 wherein said image is an X-ray image.