Abstract:
A composition for use as a contrast medium being particularly suitable for imaging of the stomach, liver, bile duct and gall bladder, said composition comprising as an active ingredient a physiologically acceptable manganese compound and an uptake promoter, wherein the uptake promoter comprises a physiologically acceptable reducing compound containing a physiologically acceptable amino acid or a salt thereof, and/or vitamin D.

Description:
The present invention relates to improvements on and relating to magnetic resonance imaging (MRI) and in particular to compositions for use as or in the preparation of MRI contrast media for imaging of the stomach, liver, bile duct and gall bladder. The MRI contrast media may also be used for imaging the pancreas and the heart. 
     MRI is now well established as a medical diagnostic tool. The ability of the technique to generate high quality images and to differentiate between soft tissues without requiring the patient to be exposed to ionizing radiation has contributed to this success. 
     Although MRI can be performed without using added contrast media, it has been found that substances which affect the nuclear spin reequilibration of the nuclei (hereinafter the &#34;imaging nuclei&#34;--generally water protons in body fluids and tissues) responsible for the magnetic resonance (MR) signals from which the images are generated may be used to enhance image contrast and, accordingly, in recent years, many such materials have been suggested as MRI contrast agents. 
     The enhanced contrast obtained with the use of contrast agents enables particular organs or tissues to be visualised more clearly by increasing or by decreasing the signal level of the particular organ or tissue relative to that of its surroundings, Contrast agents raising the signal level of the target site relative to that of its surroundings are termed &#34;positive&#34; contrast agents whilst those lowering the signal level relative to surroundings are termed &#34;negative&#34; contrast agents. 
     The majority of materials now being proposed as MRI contrast media achieve a contrast effect because they contain paramagnetic, superparamagnetic or ferromagnetic species. 
     For ferromagnetic and superparamagnetic contrast agents, which are negative MRI contrast agents, the enhanced image contrast derives primarily from the reduction in the spin reequilibration parameter known as arising from the effect on the imaging nuclei of the fields generated by the ferromagnetic or superparamagnetic particles. 
     Paramagnetic contrast agents on the other hand may be either positive or negative MRI contrast agents. The effect of paramagnetic substances on magnetic resonance signal intensities is dependent on many factors, the most important of which are the concentration of the paramagnetic substances at the imaged site, the nature of the paramagnetic substance itself, and the pulse sequence and magnetic field strength used in the imaging routine. 
     Generally, however, paramagnetic contrast agents are positive MRI contrast agents at low concentrations where their T 1  lowering effect dominates, and negative MRI contrast agents at higher concentrations where their T 2  lowering effect is dominant. In either event, the relaxation time reduction results from the effect on the imaging nuclei of the magnetic fields generated by the paramagnetic centres. 
     The use of paramagnetic, ferromagnetic and superparamagnetic materials as MRI contrast agents has been widely advocated, and broad ranges of suitable materials have been suggested in the literature. 
     An example of a physiologically acceptable paramagnetic material known for use as an MRI contrast agent is manganese ion, which may conveniently be used in the form of its salts, chelates or other complexes. Indeed, even at very low intravenuos dosages (about 5-10 μmol/kg body weight) manganese ions have been found to be particularly effective as a contrast agent for imaging of the liver. 
     However, manganese, when administered intravenously as a contrast agent, may be teratogenic at clinical dosages. Administered intravenously, manganese is also known to interfere with the normal functioning of the heart by replacement of calcium in the calcium pump of the heart. It has been reported that dosages of manganese injected into rats in excess of 0.1 mmol/kg body weight were often lethal. 
     In order to reduce the direct effect on the heart, oral administration has been proposed, This ensures passage of the contrast agent through the liver before going to the heart and thereby decreasing the possibility of a heart attack. 
     Oral administration of MnCl 2  as a liver imaging MR contrast agent has been proposed, and orally administered MnCl 2  has not been found to be teratogenic. However, the adsorption of MnCl 2  through the gut is poor, and as a result the dosage required for clinical efficacy is of the order 100-1000 μmol/kg body weight. In the event of damage to the gut resulting in increased uptake, such a high dosage level still has the potential for causing undesired adverse effects, e.g. cardiac effects. 
     The toxicity of manganese ions precludes their administration in amounts large enough to be useful in MRI. It has been found that paramagnetic chelates are less toxic than the free ions, but the chelation will prevent or reduce the enhancement of binding of paramagnetics with tissues. This means that a greater amount of the chelate will be needed to produce the same effect as the free ions. 
     EP A2 308 983 describes a MR imaging composition containing manganese(II) coordination compounds and their use in MRI. The invention is in particular directed to manganese(II) coordination complexes with water-soluble amino acids, to MR imaging compositions containing these complexes, and to their use in MRI. These compounds have proven to be more safe and provide the same relaxivity as manganese salts. The non-chelated compounds do not affect the binding of manganese to body tissue and blood components, and particularly heart and liver tissue, and reduce the toxicity. Solutions of the compound can be administered rectally, orally or parenterally, preferably parenterally. 
     Although it is stated that these solutions may be administered orally, none of the examples show what oral dosage is neccessary to achieve a measuarable MR image. Only injected dosages have been reported, and these dosages are not suffient for oral administration. 
     We have now surprisingly found that gastrointestinal tract manganese contrast agents suitable for imaging of the liver may be produced by the incorporation of an uptake promoter capable of enhancing manganese transport across the membranes of the gastrointestinal tract. 
     Compounds which have been found to be suitable for use as uptake promoters include amino acids, vitamin D or a combination thereof. 
     Thus, viewed from one aspect the present invention provides a contrast medium composition comprising a physiologically acceptable manganese compound, an uptake promoter and a physiologically acceptable carrier or excipient, wherein the uptake promoter comprises an amino acid and/or vitamin D. 
     The contrast medium composition according to the invention may comprise a manganese compound together with a mixture of several uptake promoters, i.e. a mixture of several amino acids and/or vitamin D. 
     The manganese compound, which preferably is soluble in gastrointestinal fluid, may for example be a salt, a chelate or another complex, or may be a mixture of different salts, chelates and/or complexes. Particularly preferred are metal chelates and salts in which the manganese is present as Mn(II) rather than Mn(III), since the former has a higher magnetic moment and thus is more effective as a MR contrast agent. Suitable salts are salts of inorganic anions, e.g. chlorides, bromides, iodies, flourides, sulfates, phosphates, preferably chlorides and salts of organic anions. 
     Another manganese source is manganese containing foodstuff such as blueberry juice, green tea and nuts. Also these manganese sources may be combined with an uptake promoter according to the invention. 
     The reducing nature of the uptake promoter is important since normal uptake of manganese by the gut tends to favour Mn(II) rather than Mn(III). 
     Examples of amino acids which have been found to be effective as uptake promoters in the compositions of the invention include all the native amino acids, i.e. alanine, valine, leucine, tryptophan, methionine, isoleucine, proline, phenylalanine, serine, glycine, threonine, cysteine, aspargine, glutamine, tyrosine, aspartic acid, glutamic acid, arginine, lycine and histidine. Particularly preferred as an uptake promoter in the composition of the invention are the amino acids L-aspartic acid and L-alanine. 
     The preferred vitamin is vitamin D 3 , but all subgroups of vitamin D are suitable. Vitamin D is a fat soluble vitamin and soluble in organic solvents. A preferred solvent is polyethylene glycol. 
     The increase in the uptake of manganese for various uptake promoters is demonstrated in the figures, which are MR images of the liver of rats after administering the contrast medium composition orally. 
     Using the compositions of the invention, the liver can be effectively MR imaged with a significant reduction in the dosage of manganese otherwise required when administered orally. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The figures illustrate cross section images of the body of rats in the liver region. The groups are defined in the example. 
     FIG. 1 illustrates images from rats of group 2 (top), group 5 (mid left), group 1 (control) (mid right), and group 7 (bottom). 
     The signal intensities were as follows: 
     Group 2: Average signal intensity 112 
     Group 5: Average signal intensity 134 
     Group 1: Average signal intensity 98 
     Group 7: Average signal intensity 121 
     FIG. 2 illustrates images from rats of group 4 (top), group 3 (mid left), group 1 (control) (mid right), and group 7 (bottom). 
     The signal intensities were as follows: 
     Group 4: Average signal intensity 127 
     Group 3: Average signal intensity 139 
     Group 1: Average signal intensity 106 
     Group 7: Average signal intensity 129 
     FIG. 3 illustrates images from rats of group 7 (top), group 4 (mid left), group 1 (control) (mid right), and group 2 (bottom). 
     The signal intensities were as follows: 
     Group 7: Average signal intensity 146 
     Group 4; Average signal intensity 135 
     Group 1: Average signal intensity 109 
     Group 2: Average signal intensity 101 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In the composition according to the invention, the preferred molar ratio of manganese to uptake promoter is from 1:0.2 to 1:50, preferably 1:1 to 1:20, more preferably 1:3 to 1:6, and most preferably about 1:5. 
     If desired, the uptake promoter may be present in whole or in part as the counterion to the manganese ions. 
     The composition according to the invention may be used to achieve a so-called &#34;double contrast effect&#34; by increasing the signal level from the liver whilst at the same time decreasing that from the surrounding tissues, in particular from the gut. Such an effect enables yet further enhancement of the image of the liver. 
     In a particularly preferred embodiment, the composition of the invention may be used in combination with a second contrast agent having either a positive or negative contrast effect. Preferably, the compositions of the invention are used in combination with a second contrast agent having an opposing contrast effect. This results in a &#34;double contrast effect&#34; enabling visualisation and margin definition of the liver to be particularly enhanced. 
     As mentioned above, paramagnetic materials, such as manganese ions, may act as either positive or negative MRI contrast agents depending upon a number of factors, including the concentration of the ions at the imaging site and the magnetic field strength used in the imaging procedure. At the concentrations of manganese comtemplated for use in the compositions of the invention, the manganese-containing contrast agent will in general function as a positive contrast agent. The second contrast agent is therefore conveniently negative contrast agent and may be any negative MRI contrast agent suitable for oral administration. 
     Examples of contrast agents for use in combination with the composition of the invention include iron (Fe), gadolinium (Gd) and dyprosium (Dy). 
     When using the composition of the invention to achieve a double contrast effect, it is particularly preferable to incorporate a viscosity enhancing agent which attains its full viscosity enhancing effect only after administration of the contrast medium. The contrast medium is thus able to be ingested in a relatively tolerable form while yet developing the desired viscosity at or during passage towards the site which is to be imaged. 
     The compositions of the invention are particularly suited to use, if required, after dispersion in aqueous media, for imaging of the liver. For such a purpose the composition may be administered into the gastrointestinal tract orally, rectally or via a stomach tube. 
     Thus, viewed from a further aspect the present invention provides a method of generating a magnetic resonance image of a human or non-human, preferably mammalian, animal body, which method comprises administering into the gastrointestinal tract of said body an effective amount of a contrast medium comprising a physiologically acceptable manganese compound and a physiologically acceptable reducing compound containing a physiologically acceptable amino acid or a salt thereof, and/or vitamin D, and generating a magnetic resonance image of the liver and the gastrointestinal tract of said body, 
     In a further embodiment the invention provides a method of generating a magnetic resonance image of a human or non-human animal body, which method comprises administering into the gastrointestinal tract of said body an effective amount af a composition comprising: (a) a first contrast agent comprising a physiologically acceptable manganese compound and a physiologically acceptable reducing compound containing a physiologically acceptable amino acid or a salt thereof, and/or vitamin D, together with (b) a second contrast agent and generating a magnetic resonance image of the liver and abdomen of said body. 
     It is possible to formulate the contrast medium immediately or shortly prior to administration by mixing the uptake promoter with the manganese species, Thus, in a further aspect the invention also provides a MRI contrast agent kit comprising in a first container a physiologically acceptable manganese compound, and in a second container a physiologically acceptable reducing compound containing an amino acid or a salt thereof, and/or vitamin D. 
     In a further embodiment of the invention the first container comprises a first contrast agent comprising a physiologically acceptable manganese compound together with a physiologically acceptable reducing compound containing an amino acid or a salt thereof, and/or vitamin D, and the second container comprises a second contrast agent. 
     The contrast agent composition of the invention may of course include components other than the uptake promoter, the manganese compound, for example conventional pharmaceutical formulation aids, such as wetting agents, buffers, disintegrants, binders, fillers, flavouring agents and liquid carrier media, such as sterile water, water/ethanol etc. 
     For oral administration, the pH of the composition is preferably in the acid range, e.g. 2 to 7, and while the uptake promoter may itself serve to yield a composition with this pH, buffers or pH adjusting agents may be used. 
     The contrast media may be formulated in conventional pharmaceutical administration forms, such as tablets, capsules, powders, solutions, dispersions, syrups, suppositories etc. When the contrast media can be administered orally, a patient can administer the contrast media himself 2 to 3 hours before being scanned. The patient is not obliged to stay in the hospital for several hours before being scanned. 
     The preferred dosage of the composition according to the present invention will vary according to a number of factors, such as the administration route, the age, weight and species of the subject, and the particular uptake promoter used. Conveniently, the dosage of manganese will be in the range from 5 to 500 μmol/kg body weight, preferably from 5 to 150 μmol/kg body weight, more preferably from 10 to 100 μmol/kg body weight, while the dosage of the uptake promoter will be in the range from 5 μmol to 1 mmol/kg body weight, preferably from 25 μmol to 0.5 mmol/kg body weight. 
     The following example illustrates the effectiveness of the uptake of manganese for various uptake promoters. 
     The studies were carried out on seven groups of rats with six rats in each group. MRI examination was done with a quadrature knee-coil in a 0.1 T MRI unit. Each scanning included four rats, and the examination was carried out three hours after the contrast medium was administered to the rats. If experiments are carried out with a higher tesla unit, e.g. 1.0 or 1.5 T, a larger increase in signal intensity can be expected. 
     After a 12 hour diet various contrast medium compositions were orally administered to the various groups of rats. The contrast medium was administered to the stomach of the rats via a catheter. Three hours after administration the rats were killed and placed in a tube. Thereafter the rats were scanned. 
     The following non-limiting example illustrates various compositions administered to various groups of rats. The results of the scannings are illustrated in the figures. 
     In the example the units μmol/kg and mg/kg refer to μmol/kg body weight and mg/kg body weight 
     EXAMPLE A 
     To group 1 (control) no contrast medium was administered. 
     To group 2 100 μmol/kg (19.6 mg/kg) manganese(II)chloride tetrahydrate were administered. 
     To group 3 100 μmol/kg (19.8 mg/kg) manganese(II)chloride tetrahydrate and 300 μmol/kg (42 mg/kg) L-aspartic acid were administered. 
     To group 4 100 μmol/kg (19.8 mg/kg) manganese(II)chloride tetrahydrate, 300 μmol/kg (42 mg/kg) L-aspartic acid and 0.1 mg/kg vitamin D 3  (10 μg/ml) were administered. 
     To group 5 100 μmol/kg (19.8 mg/kg) manganese(II)chloride tetrahydrate and 300 μmol/kg (25 mg/kg) L-alanine were administered. 
     To group 6 100 μmol/kg (19.8 mg/kg) manganese(II)chloride tetrahydrate, 300 μmol/kg (25 mg/kg) L-alanine and 0.1 mg/kg vitamin D 3  (10 μg/ml) were administered. 
     To group 7 100 μmol/kg (19.8 mg/kg) and 0.1 mg/kg vitamin D 3  (10 μg/ml) were administered. 
     Now referring to FIG. 1 it can be seen that the average signal intensity of the liver is substantially increased after administration orally of manganese(II)chloride tetrahydrate and L-alanine or manganese(II)chloride tetrahydrate and vitamin D 3 , respectively. 
     Referring to FIG. 2 it can be seen that the average signal intensity of the liver is substantially increased after administration orally of manganese(II)chloride tetrahydrate and L-aspartic acid and vitamin D 3 , L-aspartic acid or vitamin D 3 , respectively. 
     Referring to FIG. 3 it can be seen that the average signal intensity of the liver is substantially increased after administration of manganese(II)chloride tetrahydrate and vitamin D3 or manganese(II)chloride tetrahydrate and L-aspartic acid and vitamin D 3 , respectively. 
     The following example illustrates preferred compositions administered to a human depending on the body weight. 
     EXAMPLE B 
     
                       TABLE______________________________________          Manganese (II)   chloride  Body weight (kg) tetrahydrate (g) L-alanine (g)______________________________________ 60            1,188       1,500   70 1,386 1,750   80 1,568 2,000   90 1,782 2,250  100 1,980 2,500______________________________________ 
    
     Before being administered to the human the manganese(II) chloride tetrahydrate and L-alanine are dissolved in 250 ml of water.