Magnetic resonance imaging method and compounds for use in the method

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DESCRIPTION

Magnetic resonance imaging procedure and compounds for use in the procedure

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

Ischemia-related diseases, particularly coronary artery disease, are present in the majority of deaths in Western countries. Myocardial ischemia is a serious condition and the rapid identification and localization of myocardial ischemia is therefore highly desirable so that necessary actions, for example therapeutic or surgical treatment, can be carried out promptly before it has place irreversible myocardial damage.

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

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

Magnetic resonance imaging (MRI) has been established as a useful cardiac imaging technique. Although MRI techniques that use 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 agents 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.

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

The Mn²⁺ is a paramagnetic metal ion that competes with Ca²⁺ to enter the contracting myocardium via Ca channels²⁺ slow, leading to a significant shortening of the T1 relaxation time and therefore greater signal intensity in normal myocardial tissue. The total influx of Mn²⁺ per unit of time is increased with higher heart rate and greater force of contraction. However, much less Mn is captured in the ischemic myocardium.²⁺ due to reduction in blood flow and reduction in contractility. Hence, ischemic myocardium can be detected and distinguished from normal myocardial tissue by MR imaging using Mn.²⁺ paramagnetic as a contrast agent. Furthermore, the Mn²⁺ not a substrate for Ca²⁺ ATPase and the Na exchanger⁺/AC²⁺ during relaxation, and is therefore held in the heart for many hours. This memory effect is a drag on developing MRI research so that a patient who is administered with a contrast agent comprising Mn²⁺ perform physical exercise outside the MR imaging equipment to increase the heart rate and then follow-up imaging is carried out up to 1 hour after administration. On the contrary to Ca²⁺, Mn²⁺ it cannot induce cardiac contraction. At high doses, that is, more than 200 pmol of Mn²⁺/ kg body weight, Mn²⁺ inhibits Ca entry²⁺ to such an extent that the force of cardiac contraction fails. At clinically relevant doses, however, Mn²⁺ it has an opposite effect, that is, it actually increases the force of cardiac contraction (Kasten et al., Eur. J. Pharmacol. 253, 35, 1994) and can show cardiac toxicity. As regards the target group of patients who have to undergo MR imaging to detect ischemia and myocardial infarction such effects are, of course, undesirable.

Lauterbur and collaborators investigated Mn²⁺ in the form of manganese chloride (MnCL) as a contrast agent in animal models (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 volume I, Academic Press, New York (1978) 752-759). Significant improvement in the image of the liver and other organs, but not blood, was demonstrated despite the use of manganese chloride. However, the potential clinical utility of manganese chloride was considered to be limited due to its acute cardiac toxicity.

The toxic effects of paramagnetic metal ions are significantly reduced after complexation with a chelating agent. This can be seen as a compromise between relaxation and toxicity, see, VM Runge et al., Work in progress: potential oral and intravenous paramagnetic NMR contrast agents. RB Lauffer, Paramagnetic Metal Complexes as Water Proton Relaxation Agents for NMR Imaging: Theory and Design. Chem.

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Rev. 87 (1987) 901-927 found that a thermodynamic formation constant of approximately 10 is required^16,5 to prevent the release of free metal ions from such complexes in vivo.

US Patent 5,246,696 describes manganese or gadolinium complexes such as [[(2-hydroxytrimethylene) dinitrile] -tetraaceto] manganese (II) of disodium and [[(2-hydroxytrimethylene) dinitrile] -tetraaceto] gadolinium (III) sodium which is said to be useful for enhancing magnetic resonance imaging of body organs and tissue and to be of low toxicity. The manganese complex is reported to reduce the T1 and T2 relaxation times of the kidney, liver, spleen, pancreas and gastrointestinal tract (column 3, lines 61 to 66). Additionally in Example 8 the manganese complex is described as reducing T1 tissue particularly in 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 example, myocardial ischemia.

WO-A-99/01162 describes a myocardial ischemia detection method in humans or animals in which contrast agents comprising manganese complexes are used in conjunction with rapid imaging. Manganese ions are believed to be rapidly taken up by and retained by viable myocardial cells, whereas in reperfused infarcted tissue manganese ions are rapidly distributed throughout the tissue but are not retained in non-viable cells. Manganese ions are therefore efficiently removed from tissue despite doing so more slowly than from blood. The imaging is said to be conveniently carried out within 3 to 6 hours after injection. No further treatment of the patient such as stress treatment is cited.

US Patent 5,980,863 describes a formulation containing Mn ions²⁺ in the form of salts, for example gluconate salts, and at least 2 times the amount of Ca ions²⁺. Mn ions are noted to be toxic; however, the ion formulation is Mn with at least twice the molar ratio of Ca ions to improve safety. Formulations showing a molar ratio of Mn / Ca of 1: 8-10 are used in the examples. A composition proposed to be covered by this patent called EVP 1001 is under development.

P. Seoane et al, Proc. Intl. Soc. Magn. Reson. Med 8 (2000) 1593 and 2047 describe an Mn ion formulation designated EVP 1001 used as an MR contrast agent. A coincident injection of dobutamine, a compound that induces pharmacological stress, was injected into pigs to demonstrate the imaging effect and safety of the agent. It is also suggested that the stress and dosage of the contrast agent can be carried out outside of the equipment.

Although the disclosed contrast agents comprising non-chelated Mn ions and Ca ions appear to have good relaxant properties, there are still complications regarding their safety. The regimen of administration with infusion over a period of several minutes seems important, an accidental bolus injection or infusion rate too fast can lead to acute cardiac toxicity problems.

Calcium salts are not harmful when they enter the bloodstream. For CaCl2, the LD50 for intravenous injection in mice is 42.2 mg / kg. In comparison with this, for the well-known cardiac venom BaCl2, the corresponding LD50 dose is 19.2 mg / kg, see IB Syed et al., Toxicol. Appl. Pharmacol. 22, (1972), 150. A further disadvantage of using a large amount of calcium in the contrast agent formulation is that calcium competes with manganese for calcium channels for the penetration of divalent ions into mycocytes. This can lead to reduced efficacy, and a subsequent need to inject higher doses of contrast agent to compensate for this effect.

Another study of the myocardial memory properties of MnCl2 was described by Hu et al., Magn. Res. In Medicine 46, (2001), 884-890. A clear contrast effect could be demonstrated on MR imaging for approximately 1 hour after intravenous infusion of MnCl2 with coincident injection of the pharmacological stress agent dobutamine. The safety risk related to the use of the highly water soluble agent MnCl2 as cited above is still a problem even when MnCl2 is administered with a low infusion rate.

Therefore, the use of injections or infusions of Mn²⁺ Non-chelate, especially as a concentrated bolus injection, includes the risk of cardiac toxicity, particularly for patients who have impaired myocardial function. The Mn²⁺ it is taken up in cells in the healthy part of the myocardium and can thus develop the function of the unaffected part of the diseased heart.

Consequently, the use of free manganese ions and other paramagnetic metal ions such as gadolinium ions in the body is limited by their toxicity. Therefore, the complexation of these cations with suitable ligands and chelators is recommended, since it serves to greatly reduce their toxicity while partially maintaining their paramagnetic properties.

However, if the thermodynamic formation constant of such complexes is high, no detectable amount of free metal ions is released fast enough in vivo, and the contrast agent will not be taken up by viable tissue cells. Commercially available extracellular contrast agents such as

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Magnevist ™ and Dotarem ™ are examples of contrast agents with high stability constants and hence are unfavorable contrast agents for the detection of myocardial ischemia.

The use of physical and / or pharmacological stress increases the contrast difference between normal and ischemic myocardium by 4-5 times. It is therefore favorable to use a stress regimen that is allowed for lower doses of the contrast agent. Furthermore, a procedure that allows the contrast agent to be administered before the patient is positioned within the MRI equipment would be a preferable procedure in the clinical setting.

Thus there is a need for a contrast agent comprising a contrast-generating moiety (eg, a paramagnetic metal ion) that is taken up by viable myocytes in an amount sufficient to provide a sustained contrast effect in the state of the art. of the imaging protocol. The remaining contrast generation must remain within the myocytes for a period of time long enough to allow the patient to undergo the MRI examination procedure, that is, it must have a "memory effect". Additionally the difference in contrast enhancement between the bloodstream, ischemic myocardial tissue, and normal myocardial tissue should be sufficient to provide the delineation between ischemic and normal myocardial tissue. The contrast agent should have a safety profile that avoids the disadvantages described above for metal ions, for example non-chelated manganese ions such as manganese salts.

It has now been surprisingly found that contrast agents that can provide release of paramagnetic metal ion intermediates (the contrast-generating moiety) - such as Mn²⁺ of metal ion complexes and a chelating complexing moiety - after intravenous injection are especially useful in the diagnosis of heart disease. Such contrast agents provide an optimal compromise between sufficient efficacy and safety. Contrast agents preferably have a "memory effect", meaning that the contrast-generating moiety of the contrast agent transiently accumulates in myocardial cells providing contrast difference between normal tissue and diseased tissue. In the imaging window the contrast in the bloodstream is not significantly different from the pre-contrast in the basal bloodstream. Furthermore, no cardiotoxicity measured as significant changes in the physiological parameters in the blood was observed during the imaging procedure.

Contrast agents that exhibit a "memory effect" allow the exposure of 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 penetrate viable cells in regions of interest, eg, the myocardium, and remain in cells during the time of image data acquisition. The concentration of paramagnetic ions should be sufficient to provide Ti shortening in the image area, but should not exceed the level where cardiotoxicity begins to be a problem. The body under examination is preferably exposed to stress before the contrast agent is administered, most preferably the contrast agent is administered under peak stress and before the patient is positioned on the MRI equipment.

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

Also described in this invention is a composition comprising:

(i) at least one complex comprising an Mn ion²⁺ and a complexing residue for a human or non-human animal body, said complex presenting a thermodynamic formation constant between 10³ and 10¹⁶ and said complex showing a dissociation greater than 50% and a half-life of less than 1 minute under physiological conditions; and (ii) 0 to 2 mol of Ca²⁺ per mole of Mn²⁺ for use as a contrast agent in an MR imaging procedure to identify diseased areas comprising areas suffering from myocardial ischemia, said procedure comprising

a) administering said contrast agent to a human or non-human animal body;

b) exposing said body to a regimen of physical and / or pharmacological stress before or simultaneously with the administration of the contrast agent;

c) record MR imaging data and;

d) optionally provide MR images of an area of ​​interest.

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

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Also described in this invention is the use of compounds of formula (II)

Mn_m HPTA Z_or (II) wherein m is 1 or 2, or is 0 to 2 and Z is hydrogen or a pharmaceutically acceptable counter ion for the preparation of a contrast agent for use as an MR contrast agent in the detection of myocardial ischemia . The IUPAC nomenclature for HPTA is 1,7-dicarboxy-2,6-bis (carboxymethyl) -4-hydroxy-2,6-diaza) -heptane. Mn is Mn²⁺. Suitable pharmaceutically acceptable counter ions are, for example, cations of ammonium, substituted ammonium, alkali metal, or alkaline earth metal (eg, calcium), or anions that are derived from an inorganic or organic acid.

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

Additional aspects of the invention will be apparent from the claims and the specification.

In the MR imaging procedure the composition is used as a contrast agent, it is important that the paramagnetic metal ions of the complex are released in a controlled manner. The release of a metal ion from the complex is related to the stability constant of the complex. It has been found that a complex presenting 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 providing 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 the accumulation in intracellular myocardium of metal ions in a concentration with risk of adverse events in the body after intravenous injections such as bolus injections. Preferably, the thermodynamic formation constant k is between 10⁵ and 10¹° (log k between 5 and 10), more preferably between 10⁷ and 10^9,5 (log k between 7 and 9.5).

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

Also described in this invention are the complexes of formula (I) ^Mnrn (^P3°10⁵') n^Zo 0) where m, n and o are positive integers from 1 to 10 and Z is hydrogen or a pharmaceutically acceptable counter ion for use in an MR contrast agent. Ammonium, alkali metal, or alkaline earth metal cations, or anions derived from a suitable inorganic or organic acid. Mn is Mn²⁺. Also described in this invention is a complex of formula (I) which is manganese triphosphate (indicated MnTPP) of formula (III)

Mn²⁺ (P_toOR₁₀5-) (III)

MnTPP has a thermodynamic formation constant of 10^7,1 (Smith & Martell, Critical Stability Constants, vol. 4, Inorganic Complexes, Plenum Press, New York (1976) page 63) and is found to provide a particularly suitable in vivo manganese release rate.

Also described in this invention are complexes of formula (II)

Mn_mHPTAZ₀ (II) wherein m is 1 or 2, or is 0 to 2 and Z is hydrogen or a pharmaceutically acceptable counter ion. Suitable pharmaceutically acceptable counter ions are, for example, cations of ammonium, substituted ammonium, alkali metal, or alkaline earth metal (eg, calcium), or anions that are derived from an inorganic or organic acid. Mn is Mn²⁺.

Example complexes of formula (II) are

Mn Na₃ HPTA (IV) and Mn₂ HPTA (V) where Mn is Mn²⁺. The complex of formula (IV) has a thermodynamic formation constant k of 10⁹'¹.

When the complex used in the composition of the invention carries a general charge, it will conveniently be used in the form of a salt with a physiologically acceptable counterion, for example an ammonium, substituted ammonium, alkali metal or alkaline earth metal cation (for example , calcium), or an anion that is derived from an inorganic or organic acid. In this regard, meglumine, calcium and sodium salts are particularly preferred.

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In one embodiment, the complex used in the invention comprises Mn²⁺ and a complexing residue, said complex shows a dissociation greater than 50% and a half-life of less than 1 minute under physiological conditions. As explained above it is important for the effectiveness of the contrast agent that Mn²⁺ is released from the complex fast enough so that a relatively high concentration of Mn is present²⁺ free, which can penetrate myocytes. On the other hand, the concentration of Mn²⁺ Free should not be such that there is a risk of acute cardiac toxicity. It has now been found that complexes showing dissociation greater than 50% under physiological conditions and a half-life of less than 1 minute are preferred as they meet these criteria. A dissociation of more than 50% under physiological conditions ensures a concentration of Mn²⁺ free that is high enough to provide good efficiency. However, it is additionally favorable that Mn is released²⁺ rapidly from the complex, hence the complexes preferably used in the process of the invention have a half-life of less than 1 minute under physiological conditions.

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

The term "physiological 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 such as , for example, Zn²⁺, Faith²⁺, Cu²⁺ or Mg²⁺. In the presence of these cations transmetalation takes place which means that Mn is released²⁺ of the complex and subsequently the complexing moiety forms complexes with these endogenous cations, when the selectivity of the complexing moiety is greater for specific endogenous cations than for Mn²⁺.

There are various possible procedures for determining the% dissociation and half-life of the complexes used in the process of the invention under physiological conditions. Generally, procedures for observing and determining dissociation kinetics are known in the art, for example, various spectroscopy procedures can be used. In one embodiment a sample of the complex is mixed with blood plasma and the dissociation kinetics are followed by HPLC. The% dissociation and half-life of the complex must be calculated from the HPLC data in a manner known in the art.

In a preferred embodiment, the% dissociation and half-life of the complexes are determined by MR spectroscopy. The complex is mixed with blood plasma at a temperature between 35 and 40 ° C (= sample) and the longitudinal relaxation rates, Tri, of the sample are determined for a certain time interval at this temperature. The longitudinal relaxation, r1, of the sample is determined as a function of time according to equation (1) ri = (R1sample - R1white) / Mn concentration²⁺[mM] (1) where η is the longitudinal relaxation (s^-1mM^-1), R1sample is the relaxation rate of the sample, that is, the complex in plasma (s^-1) and R1white is the relaxation rate of the plasma without the complex (s^-1). Preferably a concentration of Mn is used²⁺ from about 0.05 to 0.2 mM as a linear interrelation that exists between R1 and the Mn concentration²⁺ in plasma for this concentration range. The% dissociation in plasma is determined according to equation (2)%^dissociated = ^{(1 - (r}1MnC12 in plasma ^{- r}1sample, t⁾ / ^r1MnCl2 in plasma⁾ * 100 (2) where r1MnC12 in plasma is the relaxation of MnCl2 in plasma and r1sample, t is the relaxation of the sample at a given time t. Preferably the time interval is about 1 hour, hence the% dissolution is calculated after 1 hour of incubation of the complex in plasma at a temperature in the range of 35 to 40 ° C. The r1 values ​​as a function versus time, for example over a 1 hour time interval, are used to calculate the dissociation rate and half-life (t1 / 2) of the complex in plasma at the given temperature.

The complexes used in the process of the invention can be produced from commercially available complexing moieties or complexing moieties described in the literature and acid salts or oxides such as paramagnetic metal acetate and chloride salts, for example, as described. described in US Patent 4,647,447. The synthesis of MnüPüP is described in EP 0290047 B1. The synthesis of HPTA complexes is described in US 5,246,696. These documents are included in this invention by reference. Briefly, the formation of the Mn complexes for use in the process of the invention involves dissolving or suspending manganese oxide or manganese salts such as manganese chloride or manganese acetate in water or a lower alcohol such as methanol, ethanol, or isopropanol. . An equimolar amount of the complexing moiety in water or a lower alcohol is added to this solution or suspension and the mixture is stirred, if necessary with heating, until the reaction is complete. If the complex formed is insoluble in the solvent used, the reaction product is conveniently isolated by filtration. If soluble the reaction product is isolated by evaporation to dryness, for example by spray drying or lyophilization.

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The above-mentioned Mn complexes used in the process of the invention comprise Mn in the form of Mn²⁺ since this ensures more effective uptake by calcium channels in myocytes. When these complexes are used in the invention, the contrast agent formulation preferably comprises an antioxidant, for example, of ascorbic acid or a reducing sugar to inhibit oxidation to Mn.³⁺ and Mn⁴⁺ with subsequent precipitation of MnO2. By providing the commercial contrast agent product in lyophilized form in an inert gas atmosphere, eg, argon gas atmosphere, the product will be stabilized during storage.

The complexes used in the invention comprise Mn²⁺ and a complexing moiety and additionally from 0.1 to 2 mol of Ca²⁺ per mole of Mn²⁺, more preferably 0.1 to 1.75 mol and most preferably 0.5 to 1 mol. Hence a particularly preferred complex is CaMnHPTA which contains 1 mole of Ca per mole of Mn²⁺ and the preferred complexing moiety HPTA. Another particularly preferred contrast agent comprises Z2MnHPTA, where Z is hydrogen or an alkali metal ion, preferably a sodium ion, and Ca²⁺ with 0.5 mol of Ca²⁺ per mole of Mn²⁺. In a preferred embodiment, the complexes used in the process of the invention are prepared from a mixture of Ca²⁺ (for example, in the form of a salt such as calcium chloride) and Mn²⁺ in the molar relationship described. In another preferred embodiment, the contrast agent is prepared by adding Ca²⁺ (for example, in the form of a salt such as calcium chloride) to a complex containing Mn²⁺ to obtain the molar ratio described.

Contrast agents comprising the complexes used in the present invention may further comprise conventional pharmaceutical or veterinary formulation aids, for example, stabilizers, antioxidants, osmorality 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, eg, water for injections. Thus, the contrast agents can be in a conventional pharmaceutical administration form such as a lyophilized product, a powder, a solution, a suspension, a dispersion, etc. However, solutions in physiologically acceptable carrier medium will generally be preferred. Suitable additives include, for example, physiologically biocompatible buffers.

Contrast agent solutions for parenteral administration, eg, intravenous administration, should be sterile and free from physiologically unacceptable agents, and should have low osmolarility to minimize irritation or other adverse effects after administration. Contrast agent solutions should preferably be isotonic or slightly hypertonic. Suitable vehicles include specifically designed aqueous vehicles used for the administration of parenteral solutions such as sodium chloride solution, Ringer's solution, dextrose solution, sodium chloride dextrose solution, lactated Ringer's solution, and other solutions. Such vehicles are described in Remington's Pharmaceutical Sciences, 15^to edition, Easton: Mack Publishing Co., pages 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th edition. Washington: American Pharmaceutical Association (1975).

Contrast agent solutions may additionally contain preservatives, antimicrobial agents, buffers, and antioxidants conventionally used for parenteral solutions. Excipients and other additives that are compatible with the complexes and that do not interfere with the manufacture, storage or use of the products can also be used.

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

In the MR imaging procedure the contrast agent is preferably administered in a bolus injection although slow injection or infusion of the contrast agent is also suitable. For Mn complexes²⁺ with constant training losses, for example in the zone of 10³ to 10⁸ there could be a risk that the release of Mn²⁺ of the complex may still be fast enough 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, eg, as a lyophilized powder, rather than the preparation of solution for injection, complexes with properties that prevent the preparation of high concentration solutions provide an additional advantage. For example, the sodium salt of MnTpP, Na3MnP310, exhibits limited solubility, thus avoiding injections of high-concentration manganese solutions. The maximum solubility of MnTPP sodium salt in water is 23 mmol / l, which is close to a formulation suitable for injection at a concentration of 15 mmol / l. At a clinical dose of 10 pmol / kg a volume of more than 45 ml of contrast agent solution needs to be injected. This injection volume further avoids rapid bolus injections of MnTPP sodium salts.

In the MRI procedure, the body is exposed to a regime of stress. Said stress is preferably physical stress outside the team, for example exercise stress, for example on a treadmill. Alternatively stress can be used, for example, with the administration of agents such as 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 tension. The use of physical or pharmacological stress significantly increases the flow

ES 2 393 780 T3 blood (4 to 5 times) which instead leads to a significant contrast difference between normal and ischemic myocardium. Additionally, a procedure that allows the contrast agent to be administered before the patient is arranged in the MRI equipment is a preferred procedure in the clinical setting.

Preferably the body undergoes MR imaging after a period of time sufficient for the MR signal intensity of the bloodstream to begin to be insignificantly different from the precontrast baseline signal intensity. More preferably, the body is subjected to MR imaging after a period of time 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 administration of the contrast agent.

Highly Ti-sensitive or ultra-fast imaging techniques are preferred that allow the generation of a series of images with as short a time interval as possible between successive images. This will ensure data acquisition during the first pass of the contrast agent through the heart, thus allowing a clinically acceptable dose of the contrast agent to be used. MRI imaging techniques capable of generating images with time intervals of less than 100 milliseconds are particularly preferred. Therefore, suitable MR imaging techniques in the method of the invention include gradient echo and echo planar imaging, especially inversion-echo planar recovery imaging, for example inversion-echo recovery imaging. planar refocused with gradient. Particularly suitable echo planar imaging techniques are those in which T1 (inversion time) is 100 to 800 milliseconds, TR (repetition time) corresponds to the heart rate and TE (echo time) is less than 20 milliseconds. , for example 10 to 20 milliseconds. The sensitivity of the imaging technique can be increased by monitoring the impulse in each heart beat. Tilt angles for use in the preparation range preceding image data acquisition can be 180 ° or 90 °, with 90 ° being preferred. The use of a 90 ° tilt angle is preferable to acquire the temporal resolution of the heartbeat.

Also described in this invention are compounds of formula (II)

Mn_m HPTA Z_or (H) where m is 1 or 2, or is 0 to 2 and Z is hydrogen or a pharmaceutically acceptable counter ion for the manufacture of a contrast agent for use as an MR contrast agent in the detection of myocardial ischemia . The IUPAC nomenclature for HPTA is 1,7-dicarboxy-2,6-bis (carboxymethyl) -4-hydroxy-2,6-diaza) -heptane. Mn is Mn²⁺. Suitable pharmaceutically acceptable counter ions are, for example, cations of ammonium, substituted ammonium, alkali metal, or alkaline earth metal (eg, calcium), or anions that are derived from an inorganic or organic acid. In a preferred embodiment Z is Na.

Particularly preferred complexes of formula (II) are

Mn Na₂ HPTA (IV) and Mn₂ HPTA (V) in which Mn is preferably Mn²⁺.

Complexes of formula (II) can be synthesized according to the procedures described in US 5,246,696.

Mn complexes for use in the present invention comprise Mn in the form of Mn²⁺ as this ensures the most effective uptake by calcium channels in myocytes. When these complexes are used in MR contrast agents, the contrast agent formulation preferably comprises an antioxidant, for example, ascorbic acid or a reducing sugar to inhibit oxidation to Mn.³⁺ and Mn⁴⁺ with subsequent precipitation of MnO2. By providing the commercial contrast agent product in lyophilized form under an inert gas atmosphere, eg argon gas atmosphere, the product will be stabilized during storage.

For MR imaging, the above-mentioned Mn complexes are preferably administered at a dose of 0.1 to 30 pmol Mn / kg body weight, more preferably 0.5 to 30 pmol Mn / kg, whichever is more. preferably 10 to 20 pmol Mn / kg.

Also described in this invention is the use of a mixture comprising a complex comprising Mn²⁺ and a complexing residue, said complex presenting a dissociation greater than 50% and a half-life of less than 1 minute under physiological conditions and from 0.1 to 2 mol of Ca²⁺ per mole of Mn²⁺ for the manufacture of a contrast agent for use in MR imaging of the myocardium, preferably for use in MR imaging of myocardial ischemia and infarction.

The present invention describes the use of a mixture comprising a complex having a thermodynamic formation constant between 10³ and 10¹⁶ comprising Mn²⁺, HPTA and 0.1 to 2 mol of Ca²⁺ per mole of Mn²⁺

ES 2 393 780 T3 for the manufacture of a contrast agent for use in MR imaging of the myocardium, preferably for use in MR imaging of myocardial ischemia and infarction.

An even more preferred embodiment is the use of a mixture comprising a complex having a

16 2+ 2+ thermodynamic formation constant between 10 and 10 comprising Mn, HPTA and 0.5 to 1 mole of Ca per mole of Mn²⁺ for the manufacture of a contrast agent for use in MR imaging of the myocardium, preferably for use in MR imaging of myocardial ischemia and infarction.

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

Examples

Example 1: Preparation of manganese complexes

a) Preparation of 15 mM MnCF (comparative example)

7.4 g (37.5 mmol) of manganese chloride tetrahydrate (MnCl₂ • 4H₂O) and 13.2 g (75 mmol) of ascorbic acid in 2.5 l of purified water giving a concentration of Mn²⁺ 15 mM. The solution was filtered through a 0.22 pm filter prior to injection.

b) MnDPDP

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

c) Preparation of 15 mM manganese triphosphate solution

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

Example 2: relaxation rate studies in pigs

An ischemic pig model was established by introducing a tube with known inner diameter (0.5 mm) and outer diameter (1.75 mm) and a length of 6 mm inserted into the LAD (left ascending coronary artery) with a catheter comprising a guide wire. After introducing the tube into the artery, the catheter and the guide wire were withdrawn. X-ray angiography was performed to verify that the coronary artery was open before and after the injection of the contrast agent, see Figure 3. These ischemic pigs weighing 20 to 30 kg were given cumulative doses of the following contrast agents: 5, 15 and 30 pmol / 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 figure 1.

Example 3: shooting

Heart rate and systolic / diastolic blood pressure were monitored in an anesthetized pig. Dobutamine was infused at a dose of 10 pg / kg body weight / minute and increased by 10 pg / kg / minute every second minute until a PRP (pressure-rhythm product) of a factor of 2.5 was reached.

10 pmol / kg body weight of MnTPP was bolted for 4 seconds. No clinical signs represented by changes in physiological parameters were observed during the experiment.

The pig was imaged at 1.5 T in a clinical scanner. Short axis view images of the myocardium were acquired 45 minutes after injection. The images were visualized in the underperfused area (see figure 2). X-ray angiography was carried out before and after the stress test imaging experiment (Figure 3).

Example 5:, calculation of the% dissociation and half-life of MnHPTA and MnHPTA containing Ca

MnHPTA preparations containing 0, 0.5 and 1 equivalent of Ca²⁺ by adding Ca-ascorbic acid to MnHPTA. Stock solutions of 200 mM manganese MnHPTA (with or without calcium) were diluted to 5 mM manganese by transferring 250 microliters of stock solution in 10 ml of RO water.

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

Four 2 ml aliquots of the blood were prepared and heated to 40 ° C. After heating, 40 microliths of 5 mM MnHPTA were added to the blood giving a sample with 0.1 mM manganese. The sample was inverted three times and the longitudinal relaxation time (T1) was determined immediately. The T1 values ​​were obtained

ES 2 393 780 T3 using a Bruker Minispec 20 MHZ (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 a sequence investment recovery with 12 different investment times. T1 values ​​were obtained every 5 minutes for a time period of one hour. The above procedure was repeated for MnHPTA samples containing 0.5 and 1 equivalent of calcium.

The dissociation in total percentage of MnHPTA after one hour was determined with equation 2. The half-life of the complex was calculated (when possible) using a validated software program (PharmNCA version 1.4, InnaPhase, Champs-sur-Marne, France). The kinetic parameters were obtained using conventional bi-exponential pharmacokinetic analysis, and the half-life of the complex was determined according to:

C (t)

<img file="ES2393780T3_D0001.tif" />

where T_1/2ayt_1/2b are the half-life of the complex of the two components and fa and fb represent the fractional volumes of the two components.

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

Example 7: Cardiovascular effects of MnHPTA with various amounts of Ca²⁺

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

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

Injections of MnHPTA (with various additions of Ca²⁺) as rapid bolus injections (injection of the full 30 pmol / kg dose over 10 seconds) through a peripheral vein in the hind paw. Saline was used as a control substance. All injections were carried out first during dobutamine stress, followed by injection at rest, that is, in the absence of dobutamine stress. Dobutamine infusion was carried out at a dose of 5-20 pg / kg / minute. The exact dose was selected by titration of the lower dose until an increase in systolic blood pressure of approximately 50% was observed. Dobutamine infusion was continued for 2 minutes after MnHPTA injection followed by 3 minutes of infusion at 30% of the selected dose. After this the dobutamine infusion was terminated.

Mean systolic, diastolic and systemic pressure (SYS, DIA MEAN), LVdP / dt, femoral artery flow, mean PAP (pulmonary arterial pressure), HR (heart rhythm) and ECG were continuously monitored and recorded with a system computer scientist.

MnHPTA injection during stress with dobutamine:

Dobutamine alone increased most of the controlled hemodynamic parameters. The most pronounced increase was observed in dP / dt max which increased 4 times. Saline injection did not cause any major change in measured hemodynamic parameters. The hemodynamic effects observed after injection of 30 pmol / kg of MnHPTA peaked within 2 minutes, that is, during maximal dobutamine infusion. The most pronounced increase in HR (approximately 50%) was observed after MnHPTA without Ca²⁺, with a simultaneous increase in femoral blood flow. The addition of Ca²⁺ attenuated these effects; 0.5 mol Ca²⁺ per mole of Mn²⁺ to an extent somewhat greater than 1 mole of Ca²⁺ per mole of Mn²⁺. Although the other parameters were affected modestly, MnHPTA with 0.5 mol of Ca²⁺ per mole of Mn²⁺ was closer to saline control in all cases compared to MnHPTA with 0 or 1 mol of Ca²⁺ per mole of Mn²⁺.

ES 2 393 780 T3

MnHPTA injection during rest:

Most of the parameters were close to saline control after MnHPTA injection. However, systemic blood pressure, particularly DIA, was temporarily reduced after injection of MnHPTA, regardless of the Ca content.²⁺. The dP / dt max increased after an increase in Ca content²⁺ of 5 MnHPTA.

Example 8: Comparison of uptake of MnHPTA containing various amounts of Ca²⁺

The comparison of uptake of MnHPTA containing 0, 1, 2 and 6 mol of Ca was studied.²⁺ per mole of Mn²⁺ in a pig model using quantitative evaluation of the R1 change in pre- and post-injection.

pigs divided into groups of 4 received MnHPTA at a dose of 15 pmol / kg body weight, MnHPTA containing 0, 1, 2 and 6 mol of Ca²⁺ per mole of Mn²⁺. R1 was assessed using a Lock-Locker sequence with 70 data points followed by signal recovery after an initial 180 ° inversion pulse in a short axis view of the myocardium. A monoexponential curve describing T1 recovery was fitted for the data and compensation for rf excitations was carried out. The R1 and AR1 in the myocardium were then calculated from the fitted T1 curves.

Higher amounts of Ca²⁺ (2 and 6 mol of Ca²⁺ per mole of Mn²⁺) resulted in significantly lower AR1s in the early phase (0 to 20 min). This can be explained by the fact that the initial uptake of Mn in the myocardium is limited because Ca competes with Mn for uptake. However, in the late phase (30 to 60 min) contrast enhancement is similar regardless of the amount of Ca.