Crystalline contrast agent for magnetic resonance imaging, kit and composition comprising it and their use

Abstract

A method of preparing a crystalline contrast agent for magnetic resonance imaging from a zwitterionic carboxylic pyridyl ligand includes mixing metal ion and the pyridyl ligand and obtaining crystals therefrom. The crystalline contrast agent includes a manganese-organic or gadolinium-organic 3D framework. The crystalline contrast agent is employed in a kit and a pharmaceutically acceptable composition. The method allows for preparing crystalline contrast agents with superior properties with easily available starting materials and with an economic and efficient process. The method allows for preparing crystalline contrast agents with exceptional water-stability and water-solubility, which exhibit high longitudinal relaxivities and with excellent stabilities under physiological conditions and low cytotoxicity. Further provided is a method for in vivo imaging of a subject, in particular a human, comprising administering the crystalline contrast agent to the subject.

Claims

1. A method of preparing a crystalline contrast agent for magnetic resonance imaging, the method comprising steps of (i) preparing a mixture comprising a manganese metal ion and a pyridyl ligand which pyridyl ligand is a zwitterionic pyridyl ligand having three carboxylic acid moieties; wherein step (i) comprises steps of a) preparing a first pre-mixture comprising steps of mixing the pyridyl ligand, a solvent and a base; and adjusting to a pH of between 5.5 and 7.5; b) preparing a second pre-mixture comprising a step of mixing a metal salt and a solvent; c) adding the second pre-mixture to the first pre-mixture to form a mixture; and d) stirring the mixture obtained in step c) for between 30 min and 90 min at a temperature of between 80 C. and 120 C.; wherein both the solvent in step a) and the solvent in step b) essentially consist of water; (ii) subjecting the obtained mixture to conditions under which crystals of the contrast agent are formed, wherein the crystals of the contrast agent crystallize in a monoclinic space group with each asymmetric unit consisting of one dissociated water and one [Mn.sub.2(Cmdcp).sub.2(H.sub.2O).sub.2] coordination entity; and (iii) separating the crystals of the contrast agent wherein the pyridyl ligand has a structure of Formula (I): ##STR00007## wherein X is a halogen and selected from Br, Cl, or I, n is an integer selected from 0, 1, 2, and 3, and wherein two of R.sup.1 to R.sup.5 are a group of Formula (II) ##STR00008## with m being an integer selected from 0, 1 and 2, and the other of R.sup.1 to R.sup.5 being hydrogen.

2. The method of claim 1, wherein the pyridyl ligand has a structure of Formula (III): ##STR00009##

3. The method of claim 2, wherein X is Br.

4. The method of claim 1, wherein the metal salt is MnCl.sub.2.

5. The method of claim 1, wherein the base is sodium hydroxide.

6. The method of claim 1, wherein step (ii) comprises steps of a) optionally stirring the mixture of step (i); and b) allowing the mixture to stand at a temperature between 20 C. and 30 C. for at least 48 hours for forming crystals of the contrast agent.

7. The method of claim 1, wherein step (iii) comprises steps of: a) separating the crystals from the mixture; and b) drying the crystals.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1A shows powder X-ray diffraction patterns of {[Mn.sub.2(Cmdcp).sub.2(H.sub.2O).sub.2](H.sub.2O)}.sub.n (compound 1) showing agreement between the simulated (I), synthesized (II), the fresh powder immersed in H.sub.2O for 24 h (Ill) and the fresh powder immersed in rats' serum for 24 h (IV).

(3) FIG. 1B shows powder X-ray diffraction patterns of {[Gd(Cmdcp)(H.sub.2O).sub.3](NO.sub.3).3H.sub.2O}.sub.n (compound 2) showing agreement between the simulated (I), synthesized (II), the fresh powder immersed in H.sub.2O for 24 h (III) and the fresh powder immersed in rats' serum for 24 h (IV).

(4) FIG. 2A illustrates the three dimensional structure of compound 1 by illustrating the coordination environment of Mn(1).

(5) FIG. 2B illustrates the coordination environment of Mn(2).

(6) FIG. 2C illustrates the coordination environment of Mn(3).

(7) FIG. 2D shows the three dimensional structure of compound 1 as viewed along the a axis and the dissociated water molecules are omitted.

(8) FIG. 2E shows the linking mode of two Cmdcp ligands in the asymmetric unit to nine different Mn centers. Color codes: Mn teal, O red, N blue, C black in FIG. 2A to 2C, gray in FIG. 2D.

(9) FIG. 3A illustrates the three dimensional structure of compound 2 by illustrating the linking of the Cmdcp ligand to five different gadolinium centers.

(10) FIG. 3B shows the coordination environment of the Gd.sup.3+ ion.

(11) FIG. 3C illustrates the monocapped square-antiprismatic coordination geometry of the Gd.sup.3+ ion.

(12) FIG. 3D illustrates the 3D structure of compound 2 as viewed along the c axis and the NO.sub.3.sup. and dissociated water molecules are omitted. Color codes: Gd teal, O red, N blue, C black in FIG. 3A to 3B, gray in FIG. 3D.

(13) FIG. 4A shows T.sub.1-weighted MRI images of compounds 1 and 2 and Gd-DTPA of varying concentrations in water (compound 1 referenced as 1 and compound 2 referenced as 2).

(14) FIG. 4B shows the r.sub.1 relaxivity curves of compounds 1 (curve 1) and 2 (curve 2) and Gd-DTPA.

(15) FIG. 5A refers to the cytotoxicity of compounds 1 and 2 by providing a diagram showing the viability of HEK293 cells incubated for 48 h with Gd-DTPA, compounds 1 (1) and 2 (2) of varying concentrations.

(16) FIG. 5B is a diagram referring to the concentration of free Mn.sup.2+ obtained at different time with concentrations of 500 M for compound 1.

(17) FIG. 5C is a diagram referring to the concentration of free Gd.sup.3+ obtained at different time with concentrations of 500 M for compound 2.

(18) FIG. 6A refers to magnetic resonance (MR) measurements with compound 1 by showing the MR signal intensity from a dynamic study of normal kidneys after intravenous administration of compound 1.

(19) FIG. 6B shows representative T.sub.1 weighted images with fast spin echo sequence from a dynamic compound 1 contrast-enhanced MR study of both normal kidneys.

(20) FIG. 6C shows the MR signal intensity from a dynamic study of normal kidneys and liver after intravenous administration of compound 1.

(21) FIG. 6D refers to 3D-SPGR and shows bilateral renal artery after 20 min intravenous administration of compound 1. RRA=right renal artery; LRA=left renal artery.

(22) FIG. 6E refers to 3D-SPGR and shows inferior vena cava (IVC) after 40 min intravenous administration of compound 1.

(23) FIG. 7A refers to the biodistribution and in vivo toxicity of compounds 1 and 2 by providing a diagram showing the ICP-MS quantification analysis of compound 1 in major organs and tissues (Br, Brain; Lu, Lung; H, Heart; Li, Liver; Sp, spleen; SI, Small intestine; Ki, Kidney; LN, Lymph Node; U, Urine; Bd, Blood) at 1 h and 24 h post-injection of compound 1.

(24) FIG. 7B shows histological morphology images of different organs of Kunming mice exposed to compounds 1 (referenced as 1) and 2 (referenced as 2) and Gd-DTPA at the concentration of 500 M for 7 days. Scale bars show 100 m.

(25) FIG. 8A refers to the results of the thermogravimetric analysis of compounds 1 and 2 by providing a curve illustrating the results of the thermogravimetric analysis of compound 1.

(26) FIG. 8B is a curve illustrating the results of the thermogravimetric analysis of compound 2.

(27) FIG. 9A shows transmission electron microscopy (TEM) image of compounds 1

(28) FIG. 9B is a TEM image of compound 2.

(29) FIGS. 10A and 10B refer to the MRI phantom measurement at 3T, with FIG. 10A showing T.sub.1 weighted MR Images of compounds 1 and 2 (compound 1 referenced as 1 and compound 2 referenced as 2) and Gd-DTPA of varying concentrations in water.

(30) FIG. 10B shows the r.sub.1 relaxivity curves of compounds 1 (curve 1) and 2 (curve 2) and Gd-DTPA. The relaxivity rates of compounds 1 and 2 and Gd-DTPA are 17.90, 13.75 and 4.98 mM.sup.1S.sup.1, respectively.

(31) FIG. 11A shows an UV-VIS absorption spectrum of compound 1 in D.I. water after the purification process with different molecular weight cut-off membranes (100 kDa, 30 kDa, 10 kDa, and 3 kDa).

(32) FIG. 11B shows an UV-VIS absorption spectrum of compound 2 in D.I. water after the purification process with different molecular weight cut-off membranes (100 kDa, 30 kDa, 10 kDa, and 3 kDa).

(33) FIG. 12 shows the MR signal intensity from a dynamic study of normal kidneys after intravenous administration of compound 1.

(34) FIG. 13A shows the pre-injection image of a dynamic contrast-enhanced MR study of normal liver after intravenous administration of compound 1

(35) FIG. 13B shows the image of FIG. 13A, after 35 min.

(36) FIG. 13C shows the image of FIG. 13A, after 60 min.

DETAILED DESCRIPTION OF THE INVENTION

(37) Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention belongs.

(38) As used herein, comprising means including the following elements but not excluding others. Essentially consisting of means that the material consists of the respective element along with usually and unavoidable impurities such as side products and components usually resulting from the respective preparation or method for obtaining the material such as traces of further components or solvents. Consisting of means that the material solely consists of, i.e. is formed by the respective element. As used herein, the forms a, an, and the, are intended to include the singular and plural forms unless the context clearly indicates otherwise. In particular the expression a metal ion and a pyridyl ligand as used in step (i) of the method for preparing the crystalline contrast agent shall mean a plurality of said metal ion and pyridyl ligand.

(39) The present invention provides a method of preparing a crystalline contrast agent for magnetic resonance imaging.

(40) Contrast agents are generally compounds which are able to alter the relaxation properties of tissues and induce an image contrast such as in magnetic resonance imaging. They are typically paramagnetic, superparamagnetic, or ferromagnetic. The extent to which a contrast agent can alter the relaxation rate is called its relaxivity which is defined as the difference in the relaxation rate of a sample measured with contrast agent and without the contrast agent. It is expressed as r.sub.1 or r.sub.2 which refers to the changes in longitudinal (1/T.sub.1) and transverse (1/T.sub.2) relaxation rate, respectively.

(41) The contrast agent for magnetic resonance imaging prepared according to the method of the present invention comprises and in particular essentially consists of a metal-organic framework (MOF). MOFs are crystalline compounds composed of two major components: a metal ion or cluster of metal ions and an organic molecule as a ligand, as mono-, di-, tri- or polydentate ligand. The metal ion(s) are coordinated to the ligand to form one-, two- or three-dimensional structures formed by repeating coordination entities extending in one, two or three dimensions. The choice of metal(s) and ligands influences the structure and properties of the MOF such as the size and shape of pores.

(42) The contrast agent prepared according to the method of the present invention is crystalline, which shall mean that the atoms or molecules are substantially organized in a structure known as a crystal. Said term is generally used in the art for any structure of ions, molecules, or atoms that are held together in an ordered arrangement. A crystalline structure is one of two types of structural ordering of atoms, ions or molecules the other being the amorphous structure which is irregular and lacks an orderly arrangement of structural units. Whether a compound is crystalline and the respective crystal system can, for example, be confirmed by means of X-ray diffraction. Preferably, the crystalline contrast agent crystallizes in a monoclinic space group.

(43) The crystalline contrast agent is prepared from a zwitterionic pyridyl carboxylate ligand. More specifically, the method of preparing the crystalline contrast agent for magnetic resonance imaging comprises steps of:

(44) (i) preparing a mixture comprising a metal ion and a pyridyl ligand which pyridyl ligand is a zwitterionic pyridyl ligand having three carboxylic acid moieties;

(45) (ii) subjecting the obtained mixture to conditions under which crystals of the contrast agent are formed;

(46) (iii) separating the crystals of the contrast agent.

(47) The term pyridyl ligand as used herein generally refers to a ligand comprising at least one optionally substituted pyridine ring. The pyridyl ligand of step (i) is a pyridyl ligand which has three carboxylic acid moieties, which means herein three free carboxylic acid functions. Those three carboxylic acid moieties can be directly or indirectly attached to the at least one pyridine ring, wherein indirectly attached means that there are methylene groups in between the carboxylic acid moieties and the pyridine ring. The pyridyl ligand is zwitterionic, i.e. is a molecule with both positive and negative electrical charges. The pyridyl ligand preferably has a structure of Formula (I):

(48) ##STR00004##
wherein X is a halogen and preferably selected from Br, Cl or I. More preferably, X is Br. n is an integer and selected from 0, 1, 2 or 3, preferably from 1 or 2 and most preferably 1. Two of R.sup.1 to R.sup.5 are a group of Formula (II):

(49) ##STR00005##
with m being an integer and selected from 0, 1 or 2 and the other of R.sup.1 to R.sup.5 being hydrogen. m is more preferably 0 or 1, most preferably 0. In preferred embodiments of the present invention, R.sub.2 and R.sub.4 are a group of Formula (II) and are preferably directly attached to carbon atoms in the pyridine ring.

(50) In particular embodiments of the present invention, the pyridyl ligand has a structure of Formula (III):

(51) ##STR00006##
with X being as defined above. More preferably, X is Br. Such pyridyl ligand can be prepared, for example, by the method described in Chen, J. X. et al. (Bent tritopic carboxylates for coordination networks: clues to the origin of self-penetration, Cryst Eng Comm. 16(2014) 7722-7730). The feature that the mixture comprises the pyridyl ligand as used herein is to be understood to cover any protonated or deprotonated form of said pyridyl ligand due to the presence of further components in the mixture added, for example, for dissolving it.

(52) The metal ion is an ion of a metal suitable for magnetic resonance imaging namely those who are able in form of a respective metal-organic framework with the pyridyl ligand to shorten the relaxation times of atoms within body tissues following administration, in particular to shorten the T.sub.1 relaxation time. Metal ions according to the present invention are in particular those with paramagnetic properties.

(53) In particular, the metal ion is a divalent or trivalent metal ion. The metal ion is preferably selected from manganese ion or gadolinium ion. The manganese ion is in particular a manganese ion in the +2 oxidation state. The gadolinium ion is in particular in the +3 oxidation state. The metal ion is preferably provided in the form of a metal salt.

(54) Step (i) of the method of the present invention preferably comprises steps of:

(55) a) preparing a first pre-mixture comprising mixing the pyridyl ligand, a solvent and a base;

(56) b) preparing a second pre-mixture comprising mixing a metal salt and a solvent; and

(57) c) adding the second pre-mixture to the first pre-mixture.

(58) The solvent in step a) preferably comprises water, more preferably essentially consists of water. The base is preferably an alkali hydroxide. Alkali hydroxides are a class of chemical compounds which are composed of an alkali metal cation, i.e. one of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), caesium (Cs), and the hydroxide anion (HO). In particular, the alkali metal cation is K or Na. More preferably, the base is NaOH, i.e. sodium hydroxide. In such embodiments, the first pre-mixture comprises the solvent of step a) and NaOH.

(59) Step a) preferably comprises mixing the pyridyl ligand and the solvent and subsequently adjusting the pH of the mixture to a pH of between 5.5 and 7.5, more preferably to a pH of about 6.0 to 7.0, by adding the base. In embodiments in which the metal ion is a manganese ion, the pH is preferably adjusted to about 7.0 by adding the base. In embodiments in which the metal ion is a gadolinium ion, the pH is preferably adjusted to about 6.0 by adding the base.

(60) The metal salt in step b) is a salt of the metal, preferably a nitrate, halogenid like chloride or bromide, sulfate, acetate, tartrate and the like of the metal. The metal salt of step b) is preferably a halogenid or a nitrate including hydrates. Preferably, the metal salt is selected from a hydrate of Gd(NO.sub.3).sub.3, in particular the hexahydrate, or MnCl.sub.2. Thus, the metal salt is most preferably selected from Gd(NO.sub.3).sub.36 H.sub.2O or MnCl.sub.2. The solvent in step b) preferably comprises and in particular essentially consists of water.

(61) Most preferably, the solvent of step a) and the solvent of step b) comprise and in particular essentially consist of water.

(62) Step (i) optionally comprises a further step d) of stirring the mixture obtained in step c) for between 30 min and 90 min, in particular for about 30 min preferably at a temperature of between 80 C. and 120 C., more preferably at about 100 C. and/or of filtering the optionally stirred mixture of step c). In embodiments of the present invention, the mixture obtained in step c) is stirred for between 30 min and 90 min, in particular for about 30 min, preferably at a temperature of between 80 C. and 120 C., more preferably at about 100 C., cooled down to a temperature of between 20 C. and 30 C., preferably to about 252 C., and then filtered, for obtaining the mixture of step (i) as the filtrate.

(63) The mixture prepared in step (i) is preferably a solution, i.e. a homogeneous mixture comprising the metal ion and the pyridyl ligand in the solvents from step a) and b) and the base, in particular both solvents essentially consist of water and the base is NaOH. In particular, the first pre-mixture prepared in step a) is a dispersion or solution comprising the pyridyl ligand in water and NaOH and the second pre-mixture prepared in step b) is a solution comprising the metal salt in water.

(64) The mixture in step (i) is preferably prepared by suspending or dissolving the pyridyl ligand in water and adjusting the pH to about 5.5 to 7.5 by means of NaOH, preparing a solution of the metal salt in water and adding said solution to the solution or dispersion comprising the pyridyl ligand.

(65) The pyridyl ligand and the metal salt are preferably used for preparing the mixture of step (i), in particular solution, in a molar ratio of the pyridyl ligand to the metal salt of between 0.8:1 and 1.8:1, in particular about 1:1 to about 1.5:1.

(66) Step (ii) of the method of the present invention preferably comprises steps of:

(67) a) optionally stirring the mixture of step (i); and

(68) b) subjecting the mixture of step (i) to a temperature of between 20 C. and 30 C. for at least 48 hours for forming crystals of the contrast agent.

(69) Step b) of step (ii) in particular comprises and is more preferably is carried out by means of allowing the mixture to stand at a temperature between 20 C. and 30 C. for at least 48 hours for forming crystals of the contrast agent. The temperature is preferably 252 C. Preferably, the mixture is allowed to stand at a temperature between 20 C. and 30 C., preferably at 252 C., for more than 48 hours for forming crystals of the contrast agent.

(70) Step (iii) of the method of the present invention preferably comprises steps of:

(71) a) separating the crystals from the mixture, preferably separating the crystals from the mixture by filtration and optionally further purifying the crystals such as by washing with a washing solvent;

(72) b) drying the crystals, preferably by vacuum drying.

(73) In an embodiment of the present invention, the crystalline contrast agent essentially consists of a crystalline manganese-organic compound and the method comprises steps of:

(74) (i) preparing a mixture comprising a manganese ion and the pyridyl ligand of Formula (III) with X being Br which step (i) comprises steps of:

(75) a) preparing a first pre-mixture comprising mixing the pyridyl ligand and water and adjusting the pH to a pH of about 7.0 by adding sodium hydroxide, preferably suspending the pyridyl ligand in water and adjusting the pH with sodium hydroxide such as 0.1 M sodium hydroxide to a pH of about 7.0, b) preparing a second pre-mixture comprising mixing MnCl.sub.2 and water; c) adding the second pre-mixture to the first pre-mixture; d) stirring the mixture obtained in step c) for between 30 min and 90 min at a temperature of between 80 C. and 120 C., cooling the mixture down to a temperature of between 20 C. and 30 C. and filtering for obtaining the mixture of step (i) as filtrate. In particular, the mixture is stirred for about 30 min at a temperature of about 100 C. and cooled down to a temperature of about 252 C., such as by allowing the stirred mixture to stand at a temperature of about 252 C. and is then filtered.
(ii) allowing the mixture to stand at a temperature between 20 C. and 30 C. for at least 48 hours for forming crystals of the manganese-organic compound. The temperature is preferably about 252 C. Preferably, the mixture is allowed to stand at a temperature between 20 C. and 30 C., in particular at about 252 C., for more than 48 hours for forming the crystals.
(iii) separating the crystals of the manganese-organic compound by filtration and then drying the crystals.

(76) In another embodiment of the present invention, the crystalline contrast agent essentially consists of a crystalline gadolinium-organic compound and the method comprises steps of:

(77) (i) preparing a mixture comprising a gadolinium ion and the pyridyl ligand of Formula (III) with X being Br, which step (i) comprises steps of:

(78) a) preparing a first pre-mixture comprising mixing the pyridyl ligand and water and adjusting the pH to a pH of about 6.0 by adding sodium hydroxide such as 0.1 M sodium hydroxide; b) preparing a second pre-mixture comprising mixing Gd(NO.sub.3).sub.36 H.sub.2O and water; c) adding the second pre-mixture to the first pre-mixture;
(ii) stirring the mixture of step (i) for between 30 min and 90 min at a temperature of between 20 C. and 30 C. and allowing the mixture after the stirring to stand at a temperature between 20 C. and 30 C. for at least 48 hours for forming crystals of the gadolinium-organic compound. The temperature is preferably about 52 C. Preferably, the mixture is allowed to stand at a temperature between 20 C. and 30 C., in particular at about 252 C., for more than 48 hours for forming the crystals.
(iii) separating the crystals of the gadolinium-organic compound by filtration and then drying the crystals.

(79) The present invention refers in a second aspect to the crystalline contrast agent obtained or obtainable by the method described above. In one embodiment, a crystalline contrast agent is provided obtained by the method described above. In another embodiment, a crystalline contrast agent is provided obtainable by the method described above.

(80) The crystalline contrast agent preferably has a molar water solubility of at least 0.1 M at a temperature of about 252 C. More preferably, the molar water solubility is at least about 0.5 M and in particular embodiments at least about 1 mM.

(81) The crystalline contrast agent preferably exhibits a longitudinal relaxivity r.sub.1 of greater than 5 mM.sup.1s.sup.1 calculated based on the molecular concentration of the contrast agent. In more preferred embodiments, the contrast agent exhibits a longitudinal relaxivity r.sub.1 of greater than 8 mM.sup.1s.sup.1, or greater than 10 mM.sup.1s.sup.1 or greater than 15 mM.sup.1s.sup.1 calculated based on the molecular concentration of the contrast agent in particular when applying a common magnetic field strengths ranging from 1.5 to 7 T (kg.Math.s.sup.2.Math.A.sup.1) such as of 3.0 T or 7.0 T. The relaxivity r.sub.1 as used herein refers to the value determined at about 30 C. in deionized water. The crystalline contrast agent obtained or obtainable with the method described above is preferably stable up to 250 C. and more preferably up to 300 C. which can be confirmed by means of thermogravimetric analysis (TGA).

(82) The metal ion release from the crystalline contrast agent is preferably less than 10 mol %, in particular less than 5 mol % after dissolving the crystalline contrast agent in water at ambient conditions such as at 252 C. for about 48 h.

(83) The crystalline contrast agent preferably comprises nanocrystals, i.e. crystals with an average diameter below 1000 nm. More preferably, the crystalline contrast agent preferably comprises crystals with an average diameter of less than about 100 nm, more preferably of less than about 90 nm. Diameter as used herein preferably refers to the Feret (or Feret's) diameter at the thickest point of a crystal. The Feret diameter is a measure of an object size along a specified direction and can be defined as the distance between the two parallel planes restricting the object perpendicular to that direction. I.e. if the Feret diameters measured for different directions differ, the diameter referred to in the present patent application always refers to the highest value measured. Average diameter refers to the average of diameter preferably measured with at least 10 crystals. The diameter is preferably measured by means of Transmission electron microscopy (TEM).

(84) In a preferred embodiment of the present invention, the crystalline contrast agent essentially consists of a manganese-organic compound comprising repeating coordination entities which can be described with the formula {[Mn.sub.2(Cmdcp).sub.2(H.sub.2O).sub.2](H.sub.2O)} extending in three dimensions (3D framework).

(85) The manganese-organic compound can in particular be described by the formula {[Mn.sub.2(Cmdcp).sub.2(H.sub.2O).sub.2](H.sub.2O)}.sub.n referenced herein as compound 1. Said compound crystallizes in the monoclinic space group P2.sub.1/c with each asymmetric unit consisting of one dissociated water and one [Mn.sub.2(Cmdcp).sub.2(H.sub.2O).sub.2] coordination entity. The two manganese ions contain one fully occupied Mn(1) and two half occupied Mn(2) and Mn(3). Two of the six carboxylate groups of two Cmdcp ligands coordinate to two Mn centers in a monodentate mode, two in bridging bidentate coordination modes to four Mn centers, and the rest two bridging carboxylate groups coordinate to three Mn centers due to two bridging O atoms from two carboxylates of two Cmdcp ligands sharing the same Mn centers. The three Mn atoms in the asymmetric unit adopt an octahedral coordination geometry.

(86) In an alternative embodiment of the present invention, the crystalline contrast agent essentially consists of a gadolinium-organic compound comprising repeating coordination entities which can be described with the formula {[Gd(Cmdcp)(H.sub.2O).sub.3](NO.sub.3).3H.sub.2O} extending in three dimensions (3D framework).

(87) The gadolinium-organic compound can in particular be described by the formula {[Gd(Cmdcp)(H.sub.2O).sub.3](NO.sub.3).3H.sub.2O}.sub.n referenced herein as compound 2. Said compound crystallizes in the monoclinic space group P2.sub.1/n and each asymmetric unit consists of one [Gd(Cmdcp)(H.sub.2O).sub.3].sup.+ coordination entity cation, one NO.sub.3.sup. anion and three dissociated water molecules. The Cmdcp ligand is located on the inversion center and coordinates to one Gd center in a chelating mode and to four Gd centers in bridging bidentate coordination modes. Each Gd center is coordinated by one chelating carboxylate and four monodentate carboxylates from five different Cmdcp ligands and three water molecules, thereby forming a monocapped square-antiprism coordination geometry.

(88) The present invention further provides a kit or a pharmaceutically acceptable composition comprising the contrast agent obtained or obtainable with the method described above and at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be, for example, a diluent or other excipient including at least one of water like water for injection, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, the kit or pharmaceutically acceptable composition may contain wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like.

(89) The kit or pharmaceutically acceptable composition can contain at least one further active ingredient. The contrast agent may also be coupled to a targeting moiety that can target a region of interest in a subject. The targeting moiety may be selected from proteins, enzymes, peptides, antibodies or the like.

(90) The pharmaceutically acceptable composition can be in either solid or liquid form. It can be a solution or suspension or a solid that is suitable for solution in, or suspension in, a diluent prior to use.

(91) In a further aspect, the present invention provides a method for in vivo imaging of a subject. The expression imaging of a subject includes imaging the whole subject or at least one part thereof like a cell, tissue or organ which is generally referred to as the site to be imaged.

(92) The method for in vivo imaging of the subject comprises:

(93) (i) administering to the subject the crystalline contrast agent obtained or obtainable by the preparation method described above;

(94) (ii) waiting a time sufficient to allow the contrast agent to accumulate at the site to be imaged; and

(95) (iii) imaging the site to be imaged with magnetic resonance imaging for obtaining one or more magnetic resonance images.

(96) The subject can be an animal or a human. Preferably, the subject is a mammal, in particular a human. The site to be imaged preferably includes one or both kidneys.

(97) The crystalline contrast agent can be administered to the subject alone or as part of a pharmaceutically acceptable composition. The relative amounts of the crystalline contrast agent of the invention, a pharmaceutically acceptable carrier, and any additional active ingredients in a pharmaceutically acceptable composition of the invention will vary, depending upon the identity, size, and condition of the subject and upon the administration route. The contrast agent may also be coupled to a targeting moiety that can target a region of interest in the subject such as proteins, enzymes, peptides, antibodies and the like.

(98) Suitable pharmaceutically acceptable compositions can be, for example, solutions or suspensions, or they may be in the form of a solid that is suitable for solution in, or suspension in, a diluent prior to administration. They may be adapted for administration by any convenient peripheral route, such as parenteral or oral administration. The crystalline contrast agent of the invention, optionally comprising other pharmaceutically active compounds, is preferably administered to the subject parenterally, in particular orally or intravenously.

(99) The amount of agent administered depends on the crystalline contrast agent, the subject and its condition and, for example, the site to be imaged and can be determined in accordance with normal clinical practice. Typically, the dosage of the contrast agent is in the range up to about 500 M, as this ensures good biocompatibility and low cytotoxicity while producing exceptional imaging properties. Dosages may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days.

(100) The crystalline contrast agent obtained or obtainable by the preparation method preferably essentially consists of a manganese-organic compound comprising repeating coordination entities which can be described with the formula {[Mn.sub.2(Cmdcp).sub.2(H.sub.2O).sub.2](H.sub.2O)} extending in three dimensions. The manganese-organic compound is in particular a manganese-organic compound which can be described by the formula {[Mn.sub.2(Cmdcp).sub.2(H.sub.2O).sub.2](H.sub.2O)}.sub.n referenced herein as compound 1.

(101) In embodiments of the present invention, step (iii) of the method for in vivo imaging of the subject comprises utilizing a contrast-enhancing imaging pulse sequence which may comprise a fast spin echo sequence or a spoiled gradient echo sequence. Preferably, said contrast-enhancing imaging pulse sequence comprises a spoiled gradient echo sequence. Such technique is known in the art, wherein manufacturers of magnetic resonance imaging equipment use different names for this technique like FLASH (fast low angle shot magnetic resonance imaging technique), SPGR (Spoiled Gradient Echo), CE-FFE-T1 (Contrast-Enhanced Fast Field Echo) or T1-FFE.

(102) In another aspect, the present invention refers to the use of the crystalline contrast agent, in particular essentially consisting of compound 1 or compound 2, or a kit or pharmaceutically acceptable composition comprising the crystalline contrast agent, in particular essentially consisting of compound 1 or compound 2, for in vivo imaging of a subject by means of magnetic resonance imaging.

EXAMPLES

(103) H.sub.3CmdcpBr was synthesized according to Chen, J. X. et al. (Bent tritopic carboxylates for coordination networks: clues to the origin of self-penetration, Cryst Eng Comm. 16(2014) 7722-7730). All the other reagents and solvents were obtained from commercial sources and used without further purification.

(104) IR spectra were recorded on a Nicolet MagNa-IR 550 infrared spectrometer. Elemental analyses for C, H, and N were performed on an EA1110 CHNS elemental analyzer. Thermogravimetric analysis (TGA) was performed on an SDTA851 Thermogravimetric Analyzer at a heating rate of 10 C. min.sup.1 under a nitrogen gas flow in an Al.sub.2O.sub.3 pan. Powder X-ray diffraction (PXRD) spectra were recorded with a Rigaku D/max-2200/PC. The X-ray generated from a sealed Cu tube was mono-chromated by a graphite crystal and collimated by a 0.5 mm MONOCAP ( Cu-K=1.54178 ). The tube voltage and current were 40 kV and 40 mA, respectively. MRI measurements were performed on a 0.5 T MRI system (SPEC-RC2, Beijing SPEC Corp.).

(105) Human embryonic kidney cell line (HEK 293) was purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China), which was routinely cultured in ATCC-formulated DMEM (Invitrogen) modified containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin streptomycin, 10,000 U mL.sup.1) in 150 mm diameter Primaria dishes at 37 C. with saturated humidity and 5% CO.sub.2. The medium was changed every 2448 h. Healthy, young, non-pregnant and nulliparous Kunming mice (2022 g) for in vivo toxicity analysis were purchased from the Laboratory Animal Center, Southern Medical University, China.

(106) The animal experiment's protocols approved by Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University were performed in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care. Female nude mice (68 weeks, 182 g, Charles River Laboratories) were used for in vivo studies.

Example 1A

Preparation of Crystalline Contrast Agents of the Present Invention

Preparation of {[Mn.SUB.2.(Cmdcp).SUB.2.(H.SUB.2.O).SUB.2.](H.SUB.2.O)}.SUB.n .(Compound 1)

(107) H.sub.3CmdcpBr (92 mg, 0.3 mmol) was suspended in H.sub.2O (25 mL) and the pH was adjusted to 7.0 with 0.1 M NaOH. Then a solution of MnCl.sub.2 (38 mg, 0.3 mmol) in H.sub.2O (20 mL) was added. The resulting mixture was stirred at 100 C. for 0.5 h, cooled to ambient temperature and then filtered. The obtained clear light yellow solution was allowed to stand at room temperature for several days. The formed yellow crystals were collected by filtration and dried in vacuum to give compound 1 (78 mg, 85%). Anal. Calcd. for C.sub.18H.sub.16Mn.sub.2N.sub.2O.sub.15: C, 35.43; H, 2.64; N, 4.59. found: C, 35.13; H, 2.74; N, 4.48. IR bands (KBr disc, cm.sup.1) v 3394 (s), 3203 (s), 3014 (s), 2947 (s), 1669 (s), 1622 (s), 1601 (s), 1446 (m), 1384 (s), 1356 (s), 1232 (w), 1174 (w), 1135 (w), 1027 (w), 912 (w), 782 (m), 768 (m), 739 (m), 727 (m), 718 (m), 628 (m), 577 (w).

Preparation of {[Gd(Cmdcp)(H.SUB.2.O).SUB.3.](NO.SUB.3.).3H.SUB.2.O}.SUB.n .(Compound 2)

(108) A solution of H.sub.3CmdcpBr (28 mg, 0.09 mmol) in H.sub.2O (5 mL) was adjusted to pH 6.0 with 0.1 M NaOH solution. Then, a solution of Gd(NO.sub.3).sub.3.6H.sub.2O (27 mg, 0.06 mmol) in H.sub.2O (1 mL) was added. The clear, colorless solution was stirred for 0.5 h and then allowed to stand at room temperature for one week. The formed colorless crystals were collected by filtration and dried in vacuum to afford compound 2 (31 mg, 56%). Anal. Calcd. for C.sub.9H.sub.17GdN.sub.2O.sub.15-2H.sub.2O: C, 21.01; H, 2.55; N, 5.45. found: C, 20.69; H, 2.01; N, 5.81. IR bands (KBr disc, cm.sup.1) v 3410 (s), 1647 (s), 1610 (s), 1390 (s), 1238 (m), 1175 (w), 1114 (w), 935 (m), 770 (m), 728 (m), 630 (m), 520 (m).

Example 1B

Characterization of the Prepared Crystalline Contrast Agents

(109) Compounds 1 and 2 obtained from the reaction of H.sub.3CmdcpBr with MnCl.sub.2 and Gd(NO.sub.3).sub.3.6H.sub.2O in the presence of NaOH are air and moisture stable under aerobic conditions. Upon ultrasonication, compounds 1 and 2 show good water-solubility with the maximum concentrations up to 2 mM for compound 1 and 500 M for compound 2, respectively. The powder X-ray diffraction (PXRD) pattern of a fresh powder of compound 1 or compound 2 immersed in H.sub.2O or rats' serum for 24 h, are in agreement with that of the simulated one, indicating their bulky phase purity and stability (PXRD, FIGS. 1A and 1B). Their bulk phase purity was further confirmed by FT-IR and elemental analyses. Thermogravimetric analysis (TGA) indicated that both compound 1 and compound 2 are stable up to 250 C. and 300 C. For compound 1, the weight loss of 8.96% from 30 C. to 272 C. corresponds to the loss of one lattice water molecule and two coordinated water molecules (calculated 8.85%). For compound 2, the weight loss of 13.17% from 30 C. to 150 C. corresponds to the loss of one lattice water molecule and three coordinated water molecules (calculated 13.21%) and the other two lattice water molecules may be lost during the drying process (FIGS. 8A and 8B). TEM micrographs for fresh compound 1 obtained from water gave the size of ca 50 nm in diameter and compound 2 is mainly composed of particles that appear silk-like shapes with 70 nm in length and 4 nm in diameter (FIGS. 9A and 9B).

Example 1C

X-Ray Crystal Structure Determinations of the Prepared Crystalline Contrast Agents

(110) Crystallographic measurements were made on a Bruker APEX II diffractometer by using graphite-monochromated Mo K (=0.71073 ) irradiation for compound 1 and compound 2. The data were corrected for Lorentz and polarization effects with the SMART suite of programs and for absorption effects with SADABS (Sheldrick, G. M., SADABS, program for empirical absorption correction of area detector data, University of Gttingen: Gttingen, Germany, 1996). All the crystal structures were solved by direct methods and refined on F.sup.2 by full-matrix least-squares techniques with SHELXTL-97 program (Sheldrick, G. M., SHELXS-97 and SHELXL-97, programs for crystal structure solution and refinement, University of Gttingen: Gttingen, Germany, 1997). For compound 1, the location of the hydrogen atoms on the coordinated and free water were suggested by Calc-OH program in WinGX suite, and the water molecules were subsequently refined as rigid groups with OH=0.85 and thermal parameters constrained to U.sub.iso(H)=1.2U.sub.eq(O). For compound 2, the hydrogen atoms on the waters were found from Fourier Map and applied OH=0.82 and U.sub.iso(H)=1.5U.sub.eq (O) for the bond length and the thermal parameters, respectively. All the non-hydrogen atoms were refined anisotropically. CCDC numbers for compound 1 and compound 2 are U.S. Pat. Nos. 1,057,253 and 1,057,254. A summary of the key crystallographic information for compound 1 and compound 2 is given in Table 1.

(111) TABLE-US-00001 TABLE 1 Crystallographic data for compounds 1 and 2 Compound 1 2 Molecular formula C.sub.18H.sub.16Mn.sub.2N.sub.2O.sub.15 C.sub.9H.sub.17GdN.sub.2O.sub.15 Formula weight 610.21 550.5 Crystal system monoclinic monoclinic Space group P2(1)/c P2(1)/n a () 7.5910(5) 10.1468(6) b () 17.6666(12) 15.5239(9) c () 15.5193(11) 10.5159(6) () 90 90 () 98.904(2) 102.2020(10) () 90 90 V (.sup.3) 2056.2(2) 1619.02(16) Z 4 4 T/K 296(2) 296(2) D.sub.calc (g cm.sup.3) 1.971 2.258 (Mo-K) () 0.71075 0.71073 (cm.sup.1) 1.32 4.185 Total reflections 20974 10257 Unique reflections 4700 2918 No. Observations 3723 2687 No. Parameters 337 280 R.sup.a 0.0527 0.0236 wR.sup.b 0.1004 0.0651 GOF.sup.c 1.112 1.108 .sub.max (e .sup.3) 0.835 1.812 .sub.min (e .sup.3) 0.565 1.374 .sup.aR = ||F.sub.o| |F.sub.c|/|F.sub.o||. .sup.bwR = {w(F.sub.o.sup.2 F.sub.c.sup.2).sup.2/w(F.sub.o.sup.2).sup.2}.sup.1/2. .sup.cGOF = {[w((F.sub.o.sup.2 F.sub.c.sup.2).sup.2)/(n p) }.sup.1/2, where n = number of reflections and p = total numbers of parameters refined. Crystal structure of {[Mn.sub.2(Cmdcp).sub.2(H.sub.2O).sub.2](H.sub.2O)}.sub.n (compound 1)

(112) Compound 1 crystallizes in the monoclinic space group P2.sub.1/c and each asymmetric unit consists of one dissociated water and one [Mn.sub.2(Cmdcp).sub.2(H.sub.2O).sub.2] molecule. The two Mn ions contain one fully occupied Mn(1) and two half occupied Mn(2) and Mn(3). Two of the six carboxylate groups of two Cmdcp ligands coordinate to two Mn centers in a monodentate fashion, two in bridging bidentate coordination modes to four Mn centers, and the rest two bridging carboxylate group coordinate to three Mn centers due to that two bridging O atoms from two carboxylates of two Cmdcp ligands sharing the same Mn centers. The two Cmdcp ligands thus act as a nine-connected node (FIG. 2E).

(113) It is notable that the three Mn atoms in the asymmetric unit adopt the same octahedral coordination geometry, but with different coordination environments. The octahedron were completed with five monodentate carboxylates and one water for Mn(1) (FIG. 2A), four monodentate carboxylates and two waters for Mn(2) (FIG. 2B), three monodentate carboxylates and three waters for Mn(3) (FIG. 2C). Therefore, the Mn(1) center acts as a five-connected node in a topological perspective, the Mn(2) center acts as a four-connected node whereas the Mn(3) center a three-connected node, accompanying with two ligands nine-connected node, leading to a 3D framework (FIG. 2D).

Crystal Structure of {[Gd(Cmdcp)(H.SUB.2.O).SUB.3.](NO.SUB.3.).3H.SUB.2.O}.SUB.n .(Compound 2)

(114) Compound 2 crystallizes in the monoclinic space group P2.sub.1/n and each asymmetric unit consists of one [Gd(Cmdcp)(H.sub.2O).sub.3].sup.+ cation, one NO.sub.3.sup. anion and three dissociated water molecules. As shown in FIG. 3A, the Cmdcp ligand is located on the inversion center and coordinates to one Gd center in a chelating fashion and to four Gd centers in bridging bidentate coordination fashion. Each Gd center is coordinated by one chelating carboxylate and four monodentate carboxylates from five different Cmdcp ligands and three water molecules, thereby forming a monocapped square-antiprism coordination geometry as shown in FIGS. 3B and 3C. The Cmdcp ligand thus acts as a five-connected node, whereas the Gd center also acts as a five-connected node, leading to a 3D framework (FIG. 3D).

Example 2

Longitudinal Relaxation Time Measurement

(115) The measurement of the longitudinal relaxation time T.sub.1 was conducted at 30 C. Five samples of compounds 1 and 2 were prepared with the concentrations of 31.25, 62.5, 125, 250, and 500 M in deionized water, respectively. Before the T.sub.1 test, these samples were ultrasonicated for 2 min to dissolve the compounds homogeneously in deionized water. The T.sub.1 of deionized water was tested as background. The T.sub.1 of the MOF solution was corrected from the background. The measurement time of each sample was ca. 2 min. Relaxivity, r.sub.1 (mM.sup.1.Math.s.sup.1), is defined as the slope of the plot of 1/T.sub.1 versus the concentration of compounds 1 and 2. T.sub.1 mapping images were acquired using an inversion recovery sequence (TE/TR=11/4000 ms) with inversion time (T.sub.I) of 200, 300, 400, 500, 600, and 700 ms. On each image, signal intensities were measured by drawing ROIs in the center of each vial. The T.sub.1 relaxation times were performed by fitting the acquired inversion recovery images to a following equation: M=M.sub.0 (12exp(T.sub.I/T.sub.1)+exp (TR/T.sub.1)), where M and M.sub.0 are measured and initial magnetization, respectively. All data fittings were performed using a nonlinear least-squares algorithm implemented in the Origin Pro 8.1 SR2 (OriginLab Co.) analysis software.

(116) The T.sub.1-weighted images and relaxivity of compounds 1 and 2 have been measured and FIG. 4A shows the T.sub.1-weighted images of compounds 1 and 2, and Gd-DTPA as a positive control, in the concentration range from 31.25 to 500 M. It is clear that the MRI signal intensity increased with the increase in their concentrations. The linear relationship between 1/T.sub.1 and the concentrations gave the relaxivity data with the r.sub.1 values being 17.50 mM.sup.1.Math.s.sup.1 for compound 1, 13.46 mM.sup.1.Math.s.sup.1 for compound 2 and 4.87 mM.sup.1.Math.s.sup.1 for Gd-DTPA, respectively (FIG. 4B). Their relaxivities at 3T are slightly higher than those at 0.5T (FIGS. 10A and 10B), indicating that the high field strength does not significantly affect their contrast enhancement. Evidently, compounds 1 and 2 exhibit much higher signal enhancement ability than Gd-DTPA. It should also be noted that both compound 1 and 2 exhibit much higher r.sub.1 relaxivities than clinically used small-molecule contrast agent OmniScan (4.1 mM.sup.1.Math.s.sup.1) (Rieter, W. J. et al., Nanoscale metal-organic frameworks as potential multimodal contrast enhancing agents, J. Am. Chem. Soc. 128(2006) 9024-9025) and reported nanoscale gadolinium MOFs [Gd.sub.2(bhc)(H.sub.2O).sub.6] (bhc=benzenehexacarboxylate, 1.5 mM.sup.1.Math.s.sup.1) (Taylor, K. M. et al., Surfactant-assisted synthesis of nanoscale gadolinium metal-organic frameworks for potential multimodal imaging, Angew Chem. Int. Ed. Engl. 47(2008) 7722-7725). This value is comparable to Gd(BDC).sub.1.5(H.sub.2O).sub.2 (BDC=1,4-benzenedicarboxylate, 20.1 mM.sup.1.Math.s.sup.1) with 1 m in length and 100 nm in diameter. These results suggest that compounds 1 and 2 are exploitable as promising MRI agents due to the presence of large payloads of paramagnetic Mn.sup.2+ and Gd.sup.3+. In addition, the good water solubility ensures accessibility of the gadolinium or manganese centers to bulk water and contributes to the r.sub.1 relaxivities and T.sub.1-weighted images.

Example 3

MTT Assay

(117) The cytotoxicity of compounds 1 and 2 was evaluated against normal human embryonic kidney cell line HEK 293 by using MTT assay. The cells were cultured in DMEM medium with 10% fetal bovine serum (FBS), 100 g/mL streptomycin and 100 U/mL penicillin at 37 C. with 5% CO.sub.2. After centrifugation at 1500g for 5 min, cell pellets were re-suspended in respective medium at the concentration of 310.sup.4 cells/mL and seeded in 96-well plates at 100 L with 310.sup.4 cells/well. Compounds 1 and 2 and Gd-DTPA (positive control) were diluted with distill water and applied in the final concentrations from 15.625 M to 500 M (four wells for each concentration per plate). Plates were incubated for 72 h, and then MTT was added to a final concentration of 0.5 mg/mL per well followed by additional incubation for 4 h. Then the reaction was stopped and the formazan dye was solubilized by adding 150 L of DMSO. The optical density was measured at 490 nm using a Bio-Rad 3500 microplate reader (Bio-Rad, Hercules, Calif., USA). Each experiment was carried out three times, and the mean values were taken. The data were reported as meanSD and all the statistical analyses were performed by SPSS11.0. The significant difference between the experimental and control groups was evaluated by T-test method and identified by P<0.05. The cell viability was calculated as follows:
Cell viability (%)=[(OD.sub.1OD.sub.3)/(OD.sub.2OD.sub.3)]100

(118) Wherein OD.sub.1, OD.sub.2 and OD.sub.3 are the optical densities of cell culture with sample, without sample and of the medium, respectively.

(119) The viability of HEK293 cells incubated with compounds 1 and 2 of varying concentrations evaluated using MTT assay (FIG. 5A) proved that there was no significant decrease in the viability of the HEK293 cells at the concentration below 500 M. At the concentration of 500 M, the cell viability was estimated to be 955% for compound 1 and 803% for compound 2. Therefore, compounds 1 and 2 showed good biocompatibility and little cytotoxicity against the model cell line when the drug concentration was below 500 M. After dissolving compounds 1 and 2 in water with concentrations of 500 M in 48 h, ICP-MS results (FIG. 5B to 5C) showed that the leakage rate is 4.2% for Mn.sup.2+ (21 M) and 7.6% for Gd.sup.3+ (38 M), respectively. Such low leakage rate may be a consequence of their low cytotoxicity.

Example 4

In Vivo MRI

(120) Female nude mice (68 weeks, 182 g, n=3, Charles River Laboratories) performed in vivo MRI was injected via the tail vein with compound 1 (300 uL, 2.7 mg Mn/kg mouse body weight), using a 7.0 T small animal MRI scanner with magnetic bore size of 310 mm, including a superconducting magnet (Magnex Scientific) with 7.0 T field strength and a gradient with 600 mT/m of maximum gradient amplitude, and 6000 T/m/s of a maximum slew rate. T1-weighted MR images of liver and kidney were acquired using fast-spin echo sequence with the following parameters: echo time (TE)/repetition time (TR): 10/750 ms, 256256 matrix, NEX=1, Field of View (FOV): 4 cm, and slice thickness: 1.0 mm. Three dimensional (3D) contrast enhanced MR angiography of aorta, renal artery and inferior vena cava was performed by using the 3D fast spoiled gradient echo (3D-FSPGR) with the following parameters: echo time (TE)/repetition time (TR): 1.7/5.3 ms, 256256 matrix, 3 NEX, field of view (FOV): 4 cm and slice thickness: 0.5 mm.

(121) Compound 1 was preferably chosen for further in vivo study because in addition to its excellent water-solubility, it showed a higher r.sub.1 relaxivity and lower cytotoxicity than compound 2. To validate the ability of compound 1 as a T.sub.1 weighted MRI agent in living subjects, MRI of nude mice has been performed in vivo (n=4) injected via the tail vein with compound 1 (300 L, 2.7 mg Mn/kg mice body weight, based on UV-Vis data, FIGS. 11A and 11B) using both 3.0 T and 7.0 T small animal MRI scanners. The coronal dynamic enhancement images of both kidneys and liver at different time points are shown in FIG. 6, FIG. 12 and FIG. 13A to 13C. After intravenous administration of compound 1, both kidneys showed remarkably positive signal enhancement after 15 minutes compared with the pre-injection images. The hyperintensity of both kidneys persisted about 240 minutes and then slightly attenuated in signal intensity after 24 h (FIG. 6A to 6C and FIG. 12), whereas the signal intensity of the liver was not increased obviously after 60 minutes (FIG. 13A to 13C). Such a significant change was attributed to the accumulation and secretion of the injected compound 1 in both kidneys. In comparison to conventional small molecule contrast agents, compound 1 remained within the vascular system for a prolonged period of time. Thus, compound 1 has potential as an MRI contrast agent for clinical use, especially in displaying the anatomy and pathology of the kidney. In addition, after intravenous administration, the utility of three-dimensional spoiled gradient recalled acquisition in steady state (3D-SPGR) imaging of kidneys provided bilateral renal artery images with superior sensitivity and diagnostic accuracy (FIG. 6D to 6E).

Example 5

In Vivo Toxicity Analysis and Biodistribution

(122) In vivo toxicity was evaluated on healthy, young, non-pregnant and nulliparous Kunming mice (2022 g). The animals were placed in clean polypropylene cages with feeding access. These cages were maintained in an air-conditioned animal house at 202 C., 50-70% relative humidity and 12 h light/dark cycle. The animals were provided with commercial mice pellet diet. All the animal procedures were conducted in compliance with the institutional ethics committee regulations and guidelines on animal welfare. After one week of acclimation, the mice were randomly divided into 4 groups, including one control group and three experimental groups with compounds 1 and 2 or Gd-DTPA. Each group consists of five females and five males, and was kept separately in polypropylene cages. Doses of 125 M, 250 M, 500 M of compounds 1 and 2 or Gd-DTPA were dissolved in deionized water. 100 L of each solution were intravenously injected through tail vein. One week later, the animals were sacrificed, and their heart, liver and kidneys were dissected out, stained with hematoxylin-eosin and examined under light microscopy.

(123) The Mn elemental analysis was performed using inductively coupled plasma mass spectrometer (ICP-MS, Thermo Scientific Xseries 2 Quadrupole). Tissues were harvested from mice (3 mice each group) for biodistribution at 1 h and 24 h after intravenous injection to quantitatively assess the biodistribution of compound 1 within various organs. The organs (no more than 500 mg) were digested in a microwave (CEM MarsXpress Microwave Digester with Teflon microwave-safe vessels) before ICP analysis The samples were suspended in freshly prepared aqua regia [trace metal grade 70% nitric acid HNO.sub.3/36% hydrochloric acid HCl (Fisher Scientific), 1:3 v/v] and heated until completely dissolved, and then diluted up to 8 mL with double-distilled water. The distribution of normal tissue and organs was expressed as a percentage of the injected dose per gram of tissue (% ID/g).

(124) The biodistribution profiles of compound 1, obtained from the ICP-MS quantitative analysis, are presented in FIG. 7A. Compound 1 displayed a significantly high level of accumulation in liver and kidney (18.91.3% ID/g, 13.01.4% ID/g), moderate in intestine and heart (8.91.2% ID/g, 7.40.1% ID/g) and low level in lung and spleen (1.40.1% ID/g, 1.70.5% ID/g). Lowest levels were observed in brain, blood and urine (0.30.01% ID/g, 0.30.01% ID/g, and 0.10.03% ID/g) at 1 h after injection. At 24 h after injection, the liver (1.290.05% ID/g), kidney (2.970.16% ID/g), spleen and intestine reached very low levels.

(125) In vivo analysis showed that the mice of each dose group retained shiny furs without symptoms of poisoning. None of them died within one week after administration. No change in body weights was observed between the treated and the control groups. As seen in FIG. 7B, the structures of organs from the exposed mice were normal, similar to those of the control group. Cardiac muscle tissue in the heart showed no hydropic degeneration. Hepatocytes in the liver appeared normal, and there were no inflammatory infiltrates. The glomerulus structure could be distinguished easily in the kidney. No necrosis was found in any of the groups.