Magnetic resonance imaging method and compounds for use in the method

0001The present invention relates to a magnetic resonance imaging (MRI) method, in particular to a MRI method enabling early detection of myocardial ischemia and to compounds for use as MR contrast agents in the method.

0002Ischemia-related diseases, in particular coronary artery diseases, account for the majority of deaths in the Western countries. Myocardial ischemia is a serious condition and rapid identification and location of myocardial ischemia is therefore highly desirable so that the necessary actions, e.g. therapeutic or surgical treatment, can be taken promptly before irreversible myocardial damage occurs.

0003Ischemic injury can be considered to result from two main events: (i) hypoxia leading to an inadequate supply of oxygen to the tissues; and (ii) decreased transport of metabolic substrates to the tissues and of metabolic end products from the tissues. Immediate consequences include energy deficit and an accumulation of protons and lactate in the region of ischemia. Other consequences include a marked, potentially harmful stimulation of the sympathetic nervous system, which ultimately leads to a rapid loss of adenosine triphosphate (ATP), an early onset of acidosis and decreased organ function.

0004Cardiac tissue, like other metabolically active tissues, is particularly vulnerable to ischemic injury. The initial phase of acute myocardial infarction is in general associated with a loss of normal contractile function, which manifests itself as regional dyskinesia. This may be due to an abrupt fall in coronary perfusion pressure, which induces an acute hibernating state, and to the rapid cessation of normal trans-membrane ion transport. Reperfusion of the ischemic myocardium prior to the onset of irreversible injury may lead to a rapid or delayed return (stunning) to normal cardiac metabolism and function.

0005Magnetic resonance imaging (MRI) has been established as a useful cardiac imaging technique. Although MR techniques using spin-echo imaging are capable of showing the anatomy of the heart, the use of contrast agents is necessary for the detection of myocardial ischemia and infarction. One class of MR contrast agent are paramagnetic contrast agents, which comprise a paramagnetic metal ion, in the form of a salt or in a complex with a chelating/complexing moiety.

0006The paramagnetic contrast agent GdDTPA (Magnevist^™) has been subject of clinical testing for use in myocardial imaging. Although this metal complex has been shown to improve identification of acute myocardial infarcts on MR images in animals and humans, its clinical use in imaging of the myocardium is limited due to its rapid excretion and distribution within the extracellular fluid space.

0007Mn²⁺ is a paramagnetic metal ion which competes with Ca²⁺ for entry in the contracting myocardium through slow Ca²⁺ channels, resulting in a significant shortening of relaxation time T₁ and thus increased signal intensity in normal myocardial tissue. The total influx of Mn²⁺ per time unit is raised during increased heart rate and force of contraction. However, in ischemic myocardium, much less Mn²⁺ is taken up because of reduction in blood flow and decrease in contractility. Hence ischemic myocardium can be detected and distinguished form normal myocardial tissue by MR imaging using paramagnetic Mn²⁺ as a contrast agent. Further, Mn²⁺ is not a substrate for Ca²⁺ ATPase and the Na⁺/Ca²⁺ exchanger during relaxation, and is hence retained in the heart for many hours. This "memory effect" lasts long enough to perform MR investigation in such a way, that a patient administered with a Mn²⁺ comprising contrast agent performs physical exercise outside the MR imager to raise heart rate and subsequent imaging is then performed up to 1 hour after administration. In contrary to Ca²⁺, Mn²⁺ can not induce heart contraction. At high doses, i.e. more than 200 µmol Mn²⁺/kg body weight, Mn²⁺ inhibits Ca²⁺ entry to such an extent, that the force of heart contraction falls. At clinically relevant doses, however, Mn²⁺ has an opposite effect, i.e. it actually increases force of heart contraction (<nplcit id="ncit0001" npl-type="s"><text>Kasten et al., Eur. J. Pharmacol. 253, 35, 1994</text></nplcit>) and may show cardiac toxicity. Regarding the target group of patients having to undergo MR imaging to detect myocardial ischemia and infarction, such effects are of course undesirable.

0008Lauterbur and coworkers investigated Mn²⁺ in the form of manganese chloride (MnCl₂ as a contrast agent in animal models (<nplcit id="ncit0002" npl-type="b"><text>P. Lauterbur et al., Augmentation of tissue water proton spin-lattice relaxation rates by in vivo addition of paramagnetic ions. In: Sutton, Leigh, Scarpa (Eds) Frontiers of Biological Energetics Vol I, Academic Press, New York (1978) 752-759</text></nplcit>). The significant image enhancement of the liver and other organs, but not blood, through the use of manganese chloride was demonstrated. However, the potential clinical utility of manganese chloride was considered to be limited due to its acute cardiac toxicity.

0009The toxic effects of paramagnetic metal ions are significantly reduced upon complex formation with a chelating agent. This may be viewed as a compromise between relaxivity and toxicity, see V. M. Runge et al., Work in progress: potential oral and intravenous paramagnetic NMR contrast agents. <nplcit id="ncit0003" npl-type="s"><text>R. B. Lauffer, Paramagnetic Metal Complexes as Water Proton Relaxation Agents for NMR Imaging: Theory and Design. Chem. Rev. 87 (1987) 901-927</text></nplcit> found that a thermodynamic formation constant of about 10^16.5 is required to prohibit release of free metal ions from such complexes <i>in vivo.</i>

0010<patcit id="pcit0001" dnum="US5246696A"><text>US patent 5,246,696</text></patcit> describes manganese or gadolinium complexes such as disodium [[(2-hydroxytrimethylene)dinitrilo]-tetraaceto]manganese (II) and sodium [[(2-hydroxytrimethylene)dinitrilo]-tetraaceto] gadolinium (III) which are said to be useful for enhancing magnetic resonance images of body organs and tissue and having low toxicity. The manganese complex is described to reduce T₁ and T₂ relaxation times of the kidney, liver, spleen, pancreas and gastrointestinal tract (col. 3, lines 61 to 66). Furthermore, in Example 8 the manganese complex is reported to reduce tissue T₁ in particularly the liver but also the heart, pancreas and kidney. However, the document does not disclose that such complexes can be used in the detection of myocardial diseases such as for instance myocardial ischemia.

0011<patcit id="pcit0002" dnum="WO9901162A"><text>WO-A-99/01162</text></patcit> describes a method of detecting myocardial ischemia in humans or animals where contrast agents comprising manganese complexes are used together with fast image generation. It is believed that manganese ions are rapidly taken up by viable myocardial cells and retained therein, whereas in reperfused infarcted tissue manganese ions rapidly distribute throughout the tissue but are not retained in non-viable cells. Manganese ions are hence efficiently cleared from this tissue albeit more slowly that from the blood. The imaging is said to be conveniently carried out within a period from 3 to 6 hours post injection. No additional treatment of the patient such as stress treatment is mentioned.

0012<patcit id="pcit0003" dnum="US5980863A"><text>US patent 5,980,863</text></patcit> describes a formulation containing Mn²⁺ ions in the form of salts, e.g. gluconate salts, and at least 2 times the amount of Ca²⁺ ions. It is noted that Mn ions are toxic; however the formulation of Mn ions with at least two times the molar ratio of Ca ions is held to improve the safety. In the examples, formulations showing a Mn/Ca molar ratio of 1 : 8-10 are used. A composition believed to be covered by this patent called EVP 1001 is in development.

0013<nplcit id="ncit0004" npl-type="s"><text>P. Seoane et al, Proc. Intl. Soc. Magn. Reson. Med 8 (2000) 1593 and 2047</text></nplcit> describe a formulation of Mn ions designated EVP 1001 used as a MR contrast agent. Coincident injection of dobutamine, a compound inducing pharmacological stress, was injected in pigs to demonstrate imaging effect and safety of the agent. It is also suggested that stress and contrast agent dosing may be performed outside the magnet.

0014Although the described contrast agents comprising non-chelated Mn ions and Ca ions appear to have good relaxivity properties, concerns regarding their safety still remain. The administration regime with infusion over a period of several minutes appears important, an accidental bolus injection or too fast infusion rate may lead to acute cardiac toxicity problems.

0015Calcium salts are not harmless when introduced into the blood circulation. For CaCl₂, the LD₅₀ on intravenous injection in mice is 42.2 mg/kg. In comparison thereto, for the well known cardiac poison BaCl₂, the corresponding LD₅₀ dose is 19.2 mg/kg, see <nplcit id="ncit0005" npl-type="s"><text>I. B. Syed et al., Toxicol. Appl. Pharmacol. 22, (1972), 150</text></nplcit>. A further disadvantage of using a large amount of calcium in the contrast agent formulation is that calcium competes with manganese for the calcium channels in the entering of the divalent ions into myocytes. This may lead to reduction of efficacy, and a subsequent need to inject higher doses of the contrast agent to compensate for this effect.

0016Another study of the myocardial memory properties of MnCl₂ was reported by<nplcit id="ncit0006" npl-type="s"><text> Hu et al., Magn. Res. in Medicine 46, (2001), 884-890</text></nplcit>. A clear contrast effect in MR imaging lasting for about 1 hour could be demonstrated after intravenous infusion of MnCl₂ with coincident injection of the pharmacological stress agent dobutamine. The safety risk related to use of the highly water-soluble agent MnCl₂ as mentioned above is still a concern even when administering MnCl₂ with a slow infusion rate.

0017Thus, the use of injections or infusions of non-chelated Mn²⁺, especially as a concentrated bolus, holds the risk of cardiac toxicity, particularly for patients having impaired myocardial function. Mn²⁺ is taken up into cells in the healthy part of the myocardium and may thus impart the function of the unaffected part of the diseased heart.

0018Consequently, the use of free manganese ions and other paramagnetic metal ions such as gadolinium ions in the body is limited by their toxicity. Thus, complexing of these cations with suitable ligands and chelates is recommended, because it serves to greatly reduce their toxicity whilst partly retaining their paramagnetic properties.

0019However, if the thermodynamic formation constant of such complexes is high, no detectable amount of free metal ion is released rapidly enough <i>in vivo,</i> and the contrast agent will not be taken up by the cells of the viable tissues. Commercially available extra cellular contrast agents such as Magnevist^™ and Dotarem^™ are examples of contrast agents with high stability constants and are hence not favourable contrast agent for the detection of myocardial ischemia.

0020The use of physical and/or pharmacological stress increases the contrast difference between normal and ischemic myocardium by 4-5 times. It is therefore favourable to use a regime of stress as it allows for lower doses of the contrast agent. Further, a method that allows the contrast agent to be administered before the patient is placed inside the MR magnet would be a preferred procedure in the clinical situation.

0021It is therefore a need for a contrast agent comprising a contrast generating moiety (e.g. a paramagnetic metal ion) that is taken up by viable myocytes in a sufficient amount to provide a sustained contrast effect in the state of art imaging protocol. The contrast generating moiety must remain inside the myocytes for a period of time sufficiently long to allow the patient to undergo the MR examination procedure, i.e. it must have a "memory effect". Further, the difference in contrast enhancement between the blood pool, the ischemic myocardial tissue and the normal myocardial tissue must be sufficient to provide delineation between the ischemic and the normal myocardial tissue. The contrast agent must have a safety profile that avoids the disadvantages described above for free metal ions, e.g. non-chelated manganese ions like manganese salts.

0022It has surprisingly now been found that contrast agents that can provide intermediate release of paramagnetic metal ions (the contrast generating moiety) - like Mn²⁺ from complexes of the metal ion and a complexing/chelating moiety - after intravenous injection are especially useful in diagnosis of cardiac diseases. Such contrast agents provide an optimal compromise between sufficient efficacy and safety. The contrast agents preferably have a "memory effect", meaning that contrast generating moiety of the contrast agent transiently accumulates in the cells of the myocardium providing contrast difference between normal tissue and diseased tissue. In the imaging window, the contrast in blood pool is not significantly different from baseline blood pool precontrast. Further, no cardiotoxicity measured as significant changes in the physiological parameters in the blood are observed during the imaging procedure.

0023The contrast agents having a "memory effect" allow exposing the human or non-human body to physical and/or pharmacological stress. Contrast agents according to the invention comprise relatively weak complexes where controlled amounts of the paramagnetic ions are released into the blood after administration. The released paramagnetic metal ions are able to enter the viable cells in the regions of interest, e.g. the myocardium, and remain in the cells during the time of image data acquisition. The concentration of the paramagnetic ions must be sufficient to provide a T₁ shorting in the imaged area, but must not exceed the level where cardiotoxicity becomes a problem. The body under examination is preferably exposed to stress before the contrast agent is administered; most preferably the contrast agent is administered at peak stress and before the patient is placed in the MR machine.

0024The present invention provides the use of a mixture comprising a complex having a thermodynamic formation constant between 10³ and 10¹⁶ which comprises Mn²⁺, HPTA and 0.1 to 2 mol Ca²⁺ per mol Mn²⁺ for the manufacture of a contrast agent for use in the MR imaging of the myocardium.

0025Also described herein is a composition comprising: <ol id="ol0001" compact="compact"><li>(i) at least one complex comprising a Mn²⁺ ion and a complexing moiety to a human or non-human animal body wherein said complex has a thermodynamic formation constant between 10³ and 10¹⁶ and wherein said complex shows a dissociation of greater than 50% and a half-life of less than 1 min under physiological conditions; and</li><li>(ii) 0 to 2 mol Ca²⁺ per mol Mn²⁺</li></ol> for use as a contrast agent in a method of MR imaging to identify diseased areas comprising areas suffering from myocardial ischemia, said method comprising <ol id="ol0002" compact="compact"><li>a) administering said contrast agent to a human or non-human animal body;</li><li>b) exposing said body to a regime of physical and/or pharmacological stress before or simultaneously to the contrast agent administration;</li><li>c) collecting MR imaging data and;</li><li>d) optionally providing MR images of an area of interest.</li></ol>

0026The contrast agent comprises either one single complex or a mixture of complexes as described in the preceding paragraph.

0027Also described herein is the use of compounds of the formula (II)         Mn_m HPTA Z_o     (II) where m is 1 or 2, o is 0 to 2 and Z is hydrogen or a pharmaceutical acceptable counterion for the manufacture of a contrast agent for use as a MR contrast agent in the detection of myocardial ischemia. The IUPAC nomenclature of HPTA is 1,7-dicarboxy-2,6-bis(carboxymethyl)-4-hydroxy-2,6-diaza)-heptane. Mn is Mn²⁺. Suitable pharmaceutical acceptable counterions are for instance ammonium, substituted ammonium, alkali metal or alkaline earth metal (e.g. calcium) cations or anions deriving from an inorganic or organic acid.

0028The compounds of formula (II) illustrate preferred examples of the complex used in the method according to the invention.

0029Further aspects of the invention will be evident from the claims and the specification.

0030In the method of MR imaging the composition is used as a contrast agent it is important that the paramagnetic metal ions from the complex are released in a controlled manner. The release of a metal ion from complex is related to the stability constant of the complex. It was found that a complex having a thermodynamic formation constant k between 10³ and 10¹⁶ (log k between 3 and 16) provides a sufficiently rapid release of the paramagnetic metal ion to provide an intracellular concentration of said metal ion in the myocardium that is adequate to generate myocardial contrast in magnetic resonance imaging. In such complexes, the release rate is sufficiently retarded to avoid intracellular myocardial accumulation of metal ions in a concentration risking adverse events in the body after intravenous injections such as bolus injections. Preferably, the thermodynamic formation constant k is between 10⁵ and 10¹⁰ (log k between 5 and 10), more preferably between 10⁷ and 10^9.5 (log k between 7 and 9.5).

0031The method of the present invention uses the complexing moiety 1,7-dicarboxy-2,6-bis(carboxymethyl)-4-hydroxy-2,6-diaza)-heptane (HPTA).

0032Also described herein are the complexes of formula (I)         Mn_m(P₃O₁₀^5-)_nZo     (I) where m, n and o are positive integers from 1 and 10 and Z is hydrogen or a pharmaceutical acceptable counterion for use in a MR contrast agent. Suitable ammonium, alkali metal or alkaline earth metal (e.g.calcium) cations or anions deriving from an inorganic or organic acid. Mn is Mn²⁺. Also described herein is a complex of formula (I) is manganese triphosphate (denoted MnTPP) of the formula (III)         Mn²⁺ (P₃O₁₀^5-)     (III) MnTPP has a thermodynamic formation constant of 10^7,1 (<nplcit id="ncit0007" npl-type="b"><text>Smith &amp; Martell, Critical Stability Constants, Vol. 4, Inorganic Complexes, Plenum Press, New York (1976) page 63</text></nplcit>) and is found to provide a particularly suitable <i>in vivo</i> release rate of manganese.

0033Also described hereih are complexes of the formula (II)         Mn_m HPTA Z_o     (II) where m is I or 2, o is 0 to 2 and Z is hydrogen or a pharmaceutical acceptable counterion. Suitable pharmaceutical acceptable counterions are for instance ammonium, substituted ammonium, alkali metal or alkaline earth metal (e.g. calcium) cations or anions deriving from an inorganic or organic acid. Mn is Mn²⁺.

0034Example complexes of the formula (II) are         Mn Na₂ HPTA (IV) and Mn₂ HPTA     (V) wherein Mn is Mn²⁺. The complex of formula (IV) has a thermodynamic formation constant k of 10^9-1.

0035Where the complex used in the composition of the invention carries an overall charge, it will conveniently be used in the form of a salt with a physiologically acceptably counterion, for example an ammonium, substituted ammonium, alkali metal or alkaline earth metal (e.g. calcium) cation or an anion deriving from an inorganic or organic acid. In this regard, meglumine, calcium and sodium salts are particularly preferred.

0036In one embodiment, the complex used in the invention comprises Mn²⁺ and a complexing moiety, said complex showing a dissociation of greater than 50% and a half-life of less than 1 min under physiologic conditions. As explained before, it is important for the efficacy of the contrast agent that Mn²⁺ is released from the complex fast enough that a relatively high concentration of free Mn²⁺ is present, which can enter the myocytes. On the other hand, the concentration of free Mn²⁺ should not be such that there is a risk of acute cardiac toxicity. It has now been found that complexes showing a dissociation of greater than 50% under physiologic conditions and a half-life of less than 1 min are preferred as they fulfil these criteria. A dissociation of greater than 50% under physiologic conditions ensures a concentration of free Mn²⁺ that is high enough to provide good efficacy. However, it is further favourable that Mn²⁺ is released quickly from the complex, hence the complexes preferably used in the method of the invention have a half-life of less than 1 min under physiologic conditions.

0037In a preferred embodiment, the complexes show a dissociation of greater than 60% under physiologic conditions, more preferably a dissociation of greater than 70% and most preferably a dissociation of greater than 80%.

0038The term "physiologic conditions" in the context of the application means in the presence of blood plasma, preferably mammalian or human blood plasma, at a temperature range of 35 to 40 °C. Blood plasma contains a variety of endogenous cations like for instance Zn²⁺, Fe²⁺, Cu²⁺ or Mg²⁺. In the presence of these cations, transmetallation occurs, meaning that Mn²⁺ is released from the complex and the complexing moiety subsequently forms complexes with these endogenous cations, when the selectivity of the complexing moiety is greater for specific endogenous cations than for Mn²⁺.

0039There are several possible methods to determine % dissociation and half-life of the complexes used in the method of the invention under physiologic conditions. Generally, methods to observe and determine dissociation kinetics are known in the art, for instance, various spectroscopic method can be used. In one embodiment, a sample of the complex is mixed with blood plasma and the dissociation kinetics are followed by HPLC. % dissociation and half-life of the complex are be calculated from the HPLC data in a way known in the art.

0040In a preferred embodiment, % dissociation and half-life of the complexes are determined by MR spectroscopy. The complex is mixed with blood plasma of a temperature between 35 to 40 °C (= sample) and the longitudinal relaxation rates, Tri, of the sample are determined over a certain time interval at this temperature. The longitudinal relaxivity, r₁, of the sample is determined as a function of time according to equitation (1) <maths id="math0001" num="(1)"><math display="block"><msub><mi mathvariant="normal">r</mi><mn mathvariant="normal">1</mn></msub><mo mathvariant="normal">=</mo><mfenced><msub><mi>Rl</mi><mi>sample</mi></msub><mo mathvariant="normal">-</mo><mi mathvariant="normal">R</mi><mo>⁢</mo><msub><mn mathvariant="normal">1</mn><mi>blank</mi></msub></mfenced><mo mathvariant="normal">/</mo><mi>concentration</mi><mspace width="1em" /><msup><mi>Mn</mi><mrow><mn mathvariant="normal">2</mn><mo mathvariant="normal">+</mo></mrow></msup><mfenced open="[" close="]"><mi>mM</mi></mfenced></math><img file="EP1572245B1_D0001.tif" /></maths> wherein r₁ is the longitudinal relaxivity (s^-1 mM^-1), R1_sample is the relaxation rate of the sample, i.e. the complex in plasma (s^-1) and R1_blank is the relaxation rate of the plasma without the complex (s^-1). Preferably, a Mn²⁺ concentration of about 0.05 to 0.2 mM is used as a linear relationship exists between R1 and the Mn²⁺ concentration in plasma for this concentration range. % dissociation in plasma is determined according to equitation (2) <maths id="math0002" num="(2)"><math display="block"><mo mathvariant="normal">%</mo><mspace width="1em" /><mi>dissociated</mi><mo mathvariant="normal">=</mo><mfenced><mn mathvariant="normal">1</mn><mo mathvariant="normal">-</mo><mfenced><msub><mi mathvariant="normal">r</mi><mrow><mn mathvariant="normal">1</mn><mo>⁢</mo><mi>MnCl</mi><mo>⁢</mo><mn mathvariant="normal">2</mn><mspace width="1em" /><mi>in plasma</mi><mo mathvariant="normal">,</mo><mi mathvariant="normal">t</mi></mrow></msub></mfenced><mo mathvariant="normal">/</mo><msub><mi mathvariant="normal">r</mi><mrow><mn mathvariant="normal">1</mn><mo>⁢</mo><mi>MnCl</mi><mo>⁢</mo><mn mathvariant="normal">2</mn><mspace width="1em" /><mi>in plasma</mi></mrow></msub></mfenced><mo mathvariant="normal">•</mo><mn mathvariant="normal">100</mn></math><img file="EP1572245B1_D0002.tif" /></maths> wherein r_{1MnC12 in plasma} is the relaxivity of MnCl₂ in plasma and r_1sample, _t is the relaxivity of the sample at a given time t. Preferably, the time interval is about 1 hour, hence % dissociation is calculated after 1 hour incubation of the complex in plasma at a temperature in the range of 35 to 40 °C. The r₁ values as a function over time, for instance over a 1 hour time interval, are used to calculate the rate of dissociation and the half-life (t_1/2) of the complex in plasma at the given temperature.

0041The complexes used in the method of the invention can be produced from commercially available complexing moieties or from completing moieties described in the literature and oxides or acid salts such as chlorine and acetate salts of the paramagnetic metal for example as described in <patcit id="pcit0004" dnum="US464744A"><text>US patent 4,647,44</text></patcit>'7. The synthesis of MnDPDP is described in <patcit id="pcit0005" dnum="EP0290047B1"><text>EP 0290047 B1</text></patcit>. The synthesis of HPTA-complexes is described in <patcit id="pcit0006" dnum="US5246696A"><text>US 5,246,696</text></patcit>. These documents are hereby included by reference. Briefly, the formation of the Mn complexes for use in the method of the invention involves dissolving or suspending manganese oxide or manganese salts like manganese chloride or manganese acetate in water or a lower alcohol like methanol, ethanol or isopropanol. To this solution or suspension is added an equimolar amount of the complexing moiety in water or a lower alcohol and the mixture is stirred, if necessary with heating, until the reaction is completed. If the complex formed is insoluble in the solvent used, the reaction product is conveniently isolated by filtering. If it is soluble, the reaction product is isolated by evaporating to dryness, e.g. by spray drying or lyophilising.

0042The aforementioned Mn complexes used in the method of the invention comprise Mn in the form of Mn²⁺ as this ensures most effective uptake by the calcium channels in the myocytes. When these complexes are used in the invention, the contrast agent formulation preferably comprises an antioxidant e.g. ascorbic acid or a reducing sugar to inhibit oxidation to Mn³⁺ and Mn⁴⁺ with subsequent precipitation of ManO₂. Providing the commercial contrast agent product in lyophilized form in an inert gas atmosphere, e.g. argon gas atmosphere, will stabilise the product during storage.

0043The complexes used in the invention comprise Mn²⁺ and a complexing moiety and further 0.1 to 2 mol Ca²⁺ per mol Mn²⁺, more preferable 0.1 to 1.75 mol and most preferably 0.5 to 1 mol. Hence, a particularly preferred complex is CaMnHPTA containing 1 mol Ca²⁺ per mol Mn²⁺ and the preferred complexing moiety HPTA. Another particularly preferred contrast agent comprises Z₂MnHPTA, wherein Z is hydrogen or an alkali metal ion, preferably a sodium ion, and Ca²+ with 0.5 mol Ca²⁺ per mol Mn²⁺. In a preferred embodiment, the complexes used in the inventive method are prepared from a mixture of Ca²⁺ (e.g. in the form of a salt like calcium chloride) and Mn²⁺ in the described molar ratio. In another preferred embodiment, the contrast agent is prepared by adding Ca²⁺ (e.g. in the form of a salt like calcium chloride) to a Mn²⁺ containing complex to obtain the described molar ratio.

0044The contrast agents comprising the complexes used in the present invention may further comprise conventional pharmaceutical or veterinary formulation aids; for example stabilisers, antioxidants, osmolality adjusting agents, buffers and pH adjusting agents. The contrast agents may be in a form suitable for injection or infusion directly or after dispersion in or dilution with a physiologically acceptable carrier medium, e.g. water for injections. Thus the contrast agents may be in a conventional pharmaceutical administration form such as a lyophilised product, a powder, a solution, a suspension, a dispersion, etc. However, solutions in physiologically acceptable carrier media will generally be preferred. Suitable additives include, for example, physiologically biocompatible buffers.

0045Solutions of the contrast agent for parenteral administration, e.g. intravenous administration, should be sterile and free from physiologically unacceptable agents, and should have low osmolality to minimize irritation or other adverse effects upon administration. The contrast agent solutions should preferably be isotonic or slightly hypertonic. Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection and other solutions. Such vehicles are described in <nplcit id="ncit0008" npl-type="b"><text>Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975</text></nplcit>) and <nplcit id="ncit0009" npl-type="b"><text>The National Formulary XIV, 14th ed. Washington: American Pharmaceutical Association (1975</text></nplcit>).

0046Solutions of the contrast agent may further contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions. Excipients and other additives which are compatible with the complexes and which will not interfere with the manufacture, storage or use of the products may also be employed.

0047Preferably, the contrast agent for use in the invention comprising the complexes is administered at a dose of 0.1 to 30 µmol paramagnetic metal ion/kg body weight, more preferably 0.5 to 30 µmol paramagnetic metal ion/kg, most preferably 10 to 20 µmol paramagnetic metal ion/kg.

0048In the MR imaging method the contrast agent is preferably administered in a bolus injection although slow injection or infusion of the contrast agent is also suitable. For Mn²⁺ complexes With low formation constants, e.g. in the area 10³ to 10⁸ there might be a risk that the release of Mn²⁺ from the complex may still be sufficiently rapid to induce cardiotoxic reactions in the body. To avoid such toxic reactions, the contrast agent comprising such complexes could be provided in solutions comprising low concentrations of such complexes. If it is preferred to provide the contrast agent in dry form, e.g. as a lyophilized powder, for on site preparation of the solution for injection, complexes with properties that avoid the preparation of high concentration solutions provide an additional advantage. For example the sodium salt of MnTPP, Na₃MnP₃O₁₀, has a limited solubility, thus prohibiting injections of highly concentrated solutions of manganese. The maximum solubility of the sodium salt of MnTPP in water is 23 mmol/L, which is close to a suitable formulation for injection at a concentration of 15 mmol/L. At a clinical dose of 10 µmol/kg, a volume of more than 45 ml of contrast agent solution needs to be injected. This injection volume additionally prohibits rapid bolus injections of sodium salts of MnTPP.

0049In the MR method, the body is exposed to a regime of stress. Said stress is preferably a physical stress outside the magnet, e.g. exercise stress for example on a treadmill. Alternatively, pharmacological stress may be employed e.g. by the administration of agents like dobutamine or dipyridamole. The contrast agent can be administered to the body during or after stress exposure. Preferably, the contrast agent is administered at peak stress. The use of physical or pharmacological stress increases blood flow significantly (4-5 times) which in turn leads to significant contrast difference between normal and ischemic myocardium. Further, a method that allows the contrast agent to be administered before the patient is placed in side the MR magnet is a preferred procedure in the clinical situation.

0050Preferably the body is subjected to MR imaging after a time period sufficient for the MR signal intensity of the blood pool to become insignificantly different from the precontrast baseline signal intensity. More preferably, the body is subjected to MR imaging after a time period of at least 5 minutes from the administration of the contrast agent, more preferably within a period of 10 to 60 minutes, even more preferably within a period of 10 to 45 minutes and most preferably within a period of 15 to 30 minutes after contrast agent administration.

0051Highly T₁-sensitive, fast or ultra-fast imaging techniques which enable the generation of a series of images with a time interval as short as possible between successive images are preferred. This will ensure the acquisition of data during the first passage of the contrast agent through the heart, thus enabling a clinically acceptable dose of contrast agent to be used. MR imaging techniques capable of generating images with time intervals of less than 100 milliseconds are particularly preferred. Thus MR imaging techniques suitable in the method of the invention include gradient echo and echo planar imaging, especially inversion recovery echo planar imaging, e.g. gradient refocused inversion recovery echo planar imaging. Particularly suitable echo planar imaging techniques are those in which TI (inversion time) is 100 to 800 milliseconds, TR (repetition time) corresponds to the heart rate and TE (echo time) is less than 20 milliseconds, e.g. 10-20 milliseconds. The sensitivity of the imaging technique may be increased by gating to every heartbeat. Flip angles for use in the preparation interval preceding image data acquisition may either be 180° or 90°, with 90° being preferred. Using a flip angle of 90° it is preferable to acquire single heart beat temporal resolution.

0052Also described herein are compounds of the formula (II)         Mn_m HPTA Z_o     (II) where m is 1 or 2, o is 0 to 2 and Z is hydrogen or a pharmaceutical acceptable counterion for the manufacture of a contrast agent for use as a MR contrast agent in the detection of myocardial ischemia. The IUPAC nomenclature of HPTA is 1,7-dicarboxy-2,6-bis(carboxymethyl)-4-hydroxy-2,6-diaza)-heptane. Mn is Mn²⁺, Suitable pharmaceutical acceptable counterions are for instance ammonium, substituted ammonium, alkali metal or alkaline earth metal (e.g. calcium) cations or anions deriving from an inorganic or organic acid. In a preferred embodiment, Z is Na.

0053Particularly preferred complexes of the formula (II) are         Mn Na₂ HPTA (IV) and Mn₂ HPTA     (V) wherein Mn is preferably Mn²⁺.

0054The complexes of the formula (II) may be synthesised according to the methods disclosed in <patcit id="pcit0007" dnum="US5246696A"><text>US 5,246,(696</text></patcit>.

0055The Mn complexes for use in the present invention comprise Mn in the form of Mn²⁺ as this ensures most effective uptake by the calcium channels in the myocytes. When these complexes are used in MR contrast agents, the contrast agent formulation preferably comprises an antioxidant e.g. ascorbic acid or a reducing sugar to inhibit oxidation to Mn³⁺ and Mn⁴⁺ with subsequent precipitation of MnO₂ Providing the commercial contrast agent product in lyophilized form in an inert gas atmosphere, e.g. argon gas atmosphere, will stabilise the product during storage.

0056For MR imaging, the aforementioned Mn complexes are preferably administered at a dose of 0.1 to 30 µmol Mn/kg body weight, more preferably 0.5 to 30 µmol Mn/kg, most preferably 10 to 20 µmol Mn/kg.

0057Also described herein is the use of a mixture comprising a complex comprising Mn²⁺ and a complexing moiety, wherein said complex shows a dissociation of greater than 50% and a half-life of less than 1 min under physiologic conditions and 0.1 to 2 mol Ca²⁺ per mol Mn²⁺ for the manufacture of a contrast agent for use in MR imaging of the myocardium, preferably for use in the MR imaging detection of myocardial ischemia and infarction.

0058The present invention describes the use of a mixture comprising a complex having a thermodynamic formation constant between 10³ and 10¹⁶ which comprises Mn²⁺, HPTA and 0.1 to 2 mol Ca²⁺ per mol Mn²⁺ for the manufacture of a contrast agent for use in the MR imaging of the myocardium, preferably for use in the MR imaging detection of myocardial ischemia and infarction.

0059An even more preferred embodiment is the use of a mixture comprising a complex having a thermodynamic formation constant between 10³ and 10¹⁶ which comprises Mn²⁺, HPTA and 0.5 to 1 mol Ca²⁺ per mol Mn²⁺ for the manufacture of a contrast agent for use in the MR imaging of the myocardium, preferably for use in the MR imaging detection of myocardial ischemia and infarction.

0060The following non-limiting examples illustrate features of the invention, R1 denotes the longitudinal relaxivity rate in s^-1.

<u>Examples</u>

<u>Example 1: Preparation of manganese complexes</u>

a) Preparation of 15 mM MnCl

<u>2</u>

(Comparison example)

00617.4 g (37.5 mmol) manganese chloride tetrahydrate (MnCl₂ • 4H₂O) and 13.2 g (75 mol) ascorbic acid were dissolved in 2.5 L of purified water to give a Mn²⁺ -concentration of 15 mM. The solution was filtrated through 0.22 µm filter prior to injection.

b) MnDPDP

0062MnDPDP is commercially available under the name Teslascan™ from Amersham Health AS, Norway.

c) Preparation of 15 mM Manganese triphosphate solution

00637.4 g (37.5 mmol) manganese chloride tetrahydrate (MnCl₂ . 4H₂O), 27.6 g (75 mmol) penta-sodium triphosphate and 13.2 g (75 mmol) ascorbic acid were dissolved in 2.5 L of purified water to give a Mn²⁺ - concentration of 15 mM. The solution was filtrated through 0.22 µm filter prior to injection.

<u>Example 2: Relaxation rate studies in pig</u>

0064An ischemia pig model was established by introduction of a tubing with known inner (0.5mm) and outer (1.75mm) diameter and a length of 6 mm inserted into LAD (left ascending coronary artery) with catheter comprising a guiding wire. After introduction of the tubing into the artery, the catheter and the guiding wire were removed. To verify that the coronary artery was open pre and post injection of the contrast agent, X-ray angiography was performed, see <figref idref="f0002">Fig 3</figref>. Three ischemic pigs of 20-30 kg body weight were given cumulative doses of the following contrast agents: 5, 15 and 30 µmol/kg of MnCl₂, MnTPP and MnDPDP. R1 was measured in blood and myocardium at 5, 15, 25 and 35 minutes after injection. The result is shown in <figref idref="f0001">Fig. 1</figref>.

<u>Example 3: Imaging</u>

0065In an anaesthesed pig, heart rate and systolic/diastolic blood pressure were monitored. Dobutamine was infused at a dose of 10 µg/kg body weight/min and increased by 10 µg/kg/min every second minute until a PRP (pressure-rate product) of a factor 2.5 was achieved.

006610 µmol/kg body weight of MnTPP was injected in a bolus during 4 seconds. No clinical signs represented by changes in physiological parameters were observed during the experiment.

0067The pig was subjected to imaging at 1.5 T in a clinical scanner. Short axis view images of the myocardium were acquired 45 minutes post injection. The images visualised an underperfused area (see <figref idref="f0002">Fig. 2</figref>). X-ray angiography was performed prior to and after the stress-test imaging experiment (<figref idref="f0002">Fig 3</figref>).

<u>Example 5: Calculation of % dissociation and half-life of MnHPTA and Ca</u>^<u>2+</u><u>containing MnHPTA</u>

0068Preparations of MnHPTA containing 0, 0.5 and 1 equivalents of Ca²⁺ by adding Ca-ascorbic acid to MnHPTA . The 200 mM manganese stock solutions of MnHPTA (with and without calcium) were diluted to 5 mM manganese by transferring 250 microlitres of the stock solution into 10-ml of RO water.

006910 ml of whole human blood was obtained from a healthy volunteer. The blood contained sodium heparin as the anti-coagulant. The percent hematocrit of the blood was determined by micro-centrifugation techniques and the concentration of endogenous metal cations determined by ICP-AES.

0070Four 2 ml aliquots of the blood were prepared and warmed to 40° C. After warming, 40 microlitres of 5 mM MnHPTA was added to the blood, resulting in sample with 0.1 mM manganese. The sample was inverted three times and the longitudinal relaxation time (T1) determined immediately. The T1 values were obtained using a 20 MHZ Bruker Minispec (Bruker Analytik GmbH, Rheinstetten, Germany) operating at 40° C. The T1 values were calculated from the mono-exponential fit of signal intensity versus time obtained from an inversion recovery sequence with 12 different inversion times. The T1 values were obtained every 5 minutes over a one hour time period. The above procedure was repeated for MnHPTA samples containing 0.5 and 1 equivalents of calcium.

0071The total percent dissociation of MnHPTA after one hour was determined form Equation 2. The complex half-lives were calculated (when possible) using a validated software program (Pharm-NCA version 1.4, InnaPhase, Champs-sur-Marne, France). The kinetic parameters were obtained using standard bi-exponential pharmacokinetic analysis, and the complex half-life was determined according to: <maths id="math0003"><math display="block"><mi>C</mi><mfenced><mi>t</mi></mfenced><mo>=</mo><msub><mi>f</mi><mi>a</mi></msub><mspace width="1em" /><mi>exp</mi><mfenced><mfrac><mrow><mo>-</mo><mn>0.693</mn><mo>⁢</mo><mi>t</mi></mrow><mrow><msub><mi>t</mi><mrow><mn>1</mn><mo>/</mo><mn>2</mn></mrow></msub><mo>⁢</mo><mi>a</mi></mrow></mfrac></mfenced><mo>+</mo><msub><mi>f</mi><mi>b</mi></msub><mspace width="1em" /><mi>exp</mi><mfenced><mfrac><mrow><mo>-</mo><mn>0.693</mn><mo>⁢</mo><mi>t</mi></mrow><mrow><msub><mi>t</mi><mrow><mn>1</mn><mo>/</mo><mn>2</mn></mrow></msub><mo>⁢</mo><mi>b</mi></mrow></mfrac></mfenced></math><img file="EP1572245B1_D0003.tif" /></maths> where <i>t</i>_<i>1</i>/<i>₂a</i> and <i>t</i>_<i>1</i>/<i>₂b</i> are the complex half-time of the two components, and <i>fa</i> and ƒb represent the fractional volumes of the two compartments.

0072The results of the study indicate that more than 80% of the MnHPTA complex is dissociated within 1 minute after exposure to whole human blood. The addition of calcium did not alter the dissociation kinetics.

<u>Example 7: Cardiovascular effects of MnHPTA with various amounts of Ca</u>^<u>2+</u>

0073The cardiovascular effects of MnHPTA with various amounts of Ca²⁺ (0, 0.5 and 1 mol Ca²⁺ per mol Mn²⁺) were investigated in the presence and absence of dobutamine induced pharmacological stress in anaesthetised dogs.

0074Anaesthesia was induced with pentobarbital (12-25 mg/kg i.v.) and fentanyl (1.5-2.5 µg/kg i.v.), followed by a continuous i.v. infusion of fentanyl (20 µg/kg/h and pentobarbital (10 mg/kg/h). Artificial ventilation was carried out by room air through a tracheal tube, aimed at achieving normal physiological blood gas values. A catheter was introduced through the right femoral artery for measurement of SAP. A microtip pressure transducer catheter (Millar) was placed into the left ventricle through the carotid artery for measurements of LVdP/dt. A Swan-Ganz catheter was introduced through the right femoral vein for measuring PAP (pulmonary arterial pressure). A 3-lead ECG was continuously monitored. An ultrasonic flow probe was placed around the left femoral artery for flow measurement. Venflon cannulas were introduced into the left and right jugular veins for contrast agent injections and dobutamine infusions, respectively.

0075Injections of MnHPTA (with various addition of Ca²⁺) were performed as rapid bolus injections (injection of the whole dose of 30µmol/kg over 10 seconds) via a peripheral forelimb vein. Saline was used as a control substance. All injections were first performed during dobutamine stress, followed by injection at rest, i.e. in the absence of dobutamine stress. Dobutamine infusion was performed at a dose of 5-20 µg/kg/min. The exact dose was be chosen by titration from the lowest dose until an increase in systolic arterial pressure by about 50% was observed. The dobutamine infusion was continued for 2 minutes after injection of MnHPTA followed by 3 min infusion at 30% of the chosen dose. The dobutamine infusion was thereafter terminated.

0076Systolic, diastolic and mean systemic arterial pressure (SYS, DIA MEAN), LVdP/dt, femoral arterial flow, mean PAP (pulmonary arterial pressure), HR (heart rate) and ECG were continuously monitored and stored by a computer system.

MnHPTA injection during dobutamine stress:

0077Dobutamine alone increased most of the monitored haemodynamic parameters. The most pronounced increase was seen in dP/dt max that increased 4 times. Saline injection did not cause any major changes in the measured haemodynamic parameters. The haemodynamic effects seen upon injection of 30 µmol/kg MnHPTA peaked within 2 minutes, i.e., during maximal dobutamine infusion. The most pronounced increase in HR (about 50%) was seen after MnHPTA without Ca²⁺, with a simultaneous increase in femoral blood flow. Addition of Ca²⁺ attenuated these effects; 0.5 mol Ca²⁺ per mol Mn²⁺ to a somewhat larger extent than 1 mol Ca²⁺ per mol Mn²⁺. Although the other parameters were affected modestly, MnHPTA with 0.5 mol Ca²⁺ per mol Mn²⁺ was closer to the saline control in all instances compared to MNHPTA with 0 or 1 mol Ca²⁺ per mol Mn²⁺.

MnHPTA injection during rest:

0078Most parameters stayed close to the saline control after injection of MnHPTA. However, systemic blood pressure, in particular DIA was transiently lowered upon MnHPTA injection, independently on the Ca²⁺ content. The dP/dt max increased upon increase in the Ca²⁺ content of MnHPTA.

<u>Example 8: Comparison of uptake of MnHPTA containing various amounts of Ca</u>^<u>2+</u>

0079Comparison of uptake of MnHPTA containing 0, 1, 2 and 6 mol Ca²⁺ per mol Mn²⁺ was studied in a pig model by using quantitative assessment of the R1 change in pre and post injection.

008012 pigs divided into 4 groups received MnHPTA in a dose of 15 µmol/kg body weight, the MnHPTA containing 0, 1, 2 and 6 mol Ca²⁺ per mol Mn²⁺. R1 was assessed using a Lock-Locker sequence with 70 data points following the signal recovery after an initial 180° inversion pulse in a short axis view of the myocardium. A monoexponential curve describing the T1 recovery was fitted to the data and compensation of the rf-excitations was performed. The R1 and ΔR1 in the myocardium was then calculated from the fitted T1 curves.

0081Higher amounts of Ca²⁺ (2 and 6 mol Ca²⁺ per mol Mn²⁺) resulted in significantly lower ΔR1 at the early phase (0 to 20 min). This can be explained by the fact that the initial uptake of Mn into the myocardium is limited due to Ca competing with Mn for the uptake. However, at the late phase (30 to 60 min) contrast enhancement is similar regardless the amount of Ca.