Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations

Definitions

• the invention relates to compositions and methods for enhancing magnetic resonance images of tissues, systems and organs.
• Magnetic resonance imaging enables noninvasive visualization of tissues and organs of the human body. Contrast in the images generated may be enhanced through the use of an agent that alters the relaxation of water in the tissues of interest relative to bulk water. Species with unpaired electrons, such as the paramagnetic transition and lanthanide metal ions, may be used for this purpose.
• Manganese chloride was investigated as a contrast agent by Lauterbur and by Wolf in animal models. Both investigators demonstrated significant image enhancement of the liver and other organs (but not blood) through the use of manganese chloride, but determined that the potential clinical utility of the agent was limited by acute cardiac toxicity. Development of contrast agents based on other paramagnetic metal ions is similarly constrained by toxicity and solubility. For instance, gadolinium chloride, acetate and sulfate demonstrate poor tolerability, including symptoms of heavy metal poisoning and accumulation of gadolinium in the liver, spleen and bone.
• the free chelator may introduce additional toxicity on its own or through chelation of those endogenous metal ions that are required as cofactors for essential enzymes or for other biological functions.
• chelates also demonstrate reduced solution relaxivity relative to the free metal ions.
• chelation provides safety at the price of reduced imaging efficacy (compared to the free metal ion). In practice this loss of efficacy may be as high as 60 -80%.
• Manganese chelate image enhancement agents are known: e.g MnDPDP, MnDTPA, MnEDTA and derivatives, Mn porphyrins such as MnTPPS 4 , and fatty acyl DTPA derivatives. These manganese chelates are not known to bind to endogenous macromolecules, as is the case for manganese ion. As a consequence, the enhanced efficacy seen for Mn ion following macromolecular association is seen for Mn chelates only as a function of the rate at which and the extent to which the manganese ion dissociates from the complex. This results in the need for increased dosing of Mn chelates relative to free Mn ion.
• the dose must be additionally increased to make up for losses due to renal excretion of the chelate during the time course of the diagnostic examination.
• Quay European patent application 308983
• This application also discusses the addition of calcium ions to the manganese amino acid solutions at levels up to 0.75 mole equivalents relative to manganese.
• Schaefer et al investigated a mixture of Mn++ and Ca++ salts in the form of manganese gluconate and calcium gluconate in a one to one mole ratio, administered intravenously to dogs, for cardiac perflision imaging. Although the agent discriminated normally perfused from ischemic tissue, Schaefer et al also noted acute cardiotoxicity similar to that seen with manganese chloride alone. The authors suggested that a possible way around the observed adverse cardiac effects might be to employ a chelate rather than a salt of manganese. No further studies employing manganese gluconate and calcium gluconate or other salts or complexes providing Mn++ and Ca++ in a higher ratio than one- to-one have subsequently appeared..
• US patents 5,525,326 and 5,716,598 describe oral manganese formulations for imaging of the gastrointestinal tract and for liver imaging; the latter takes advantage of the fact that the blood supply from the GI tract passes through the liver, which removes the manganese from the blood stream prior to return of the blood to the heart .
• Additional oral agents have been investigated, including manganese polymers, manganese impregnated molecular sieves, manganese clays and foodstuffs with high manganese content, such as blueberry juice .
• cardiovascular safety is achieved for these agents at the expense of limiting the diagnostic utility of the agents to MR examination of the GI tract and in some cases, the liver.
• the administration of manganese in nanop articulate form has been described in US patent 5,401,492.
• particulate approaches include sequestration of Mn compounds in liposomes and metal clusters, such as manganese oxalate and manganese hydroxyapatite.
• Particulate agents are useful for a limited number of diagnostic applications, namely, the gastrointestinal tract and organs, such as the liver and the spleen, that are involved in the uptake and sequestration of blood borne particles.
• the gastrointestinal tract and organs such as the liver and the spleen
• an agent that could be employed for a wide variety of tissues, systems and organs that are physiologically remote from the gastrointestinal tract are physiologically remote from the gastrointestinal tract.
• a diagnostic composition comprising a source of a diagnostically effective quantity of Mn++ ion , a source of Ca++ ion and a pharmaceutically acceptable carrier for parenteral administration.
• the Ca++ ion is present in a molar ratio of from 2: 1 to 40: 1 with respect to the Mn++ ion.
• Preferred sources of Mn++ are manganese salts, such as manganese acetate, chloride, gluconate, gluceptate, lactate and sulfate or mixtures thereof.
• Manganese gluconate or manganese gluceptate are most preferred sources of Mn++.
• Preferred sources of Ca++ are calcium salts, such as calcium acetate, chloride, gluconate, gluceptate, and lactate or mixtures thereof. Calcium gluconate and calcium gluceptate are most preferred sources of Ca++.
• the molar ratio of calcium to manganese is preferably from 4: 1 to 20: 1 and most preferably from 8: 1 to 10: 1.
• One embodiment of the composition aspect of the invention is a unit dosage form comprising a salt of manganese containing from 5 mg to 200 mg of manganese, a salt of calcium containing from 20 mg to 3g of calcium, and a vehicle suitable for parenteral injection.
• the invention in another aspect, relates to a method for enhancing a magnetic resonance image of a mammalian tissue, organ or system.
• the method comprises administering to a mammal a diagnostically effective amount of a source of Mn++ ion together with from 2 to 200 molar equivalents of a source of Ca++ ions.
• the preferred sources of Mn++ and Ca++ are as before.
• the sources of manganese and calcium may be administered intravenously at 1 ⁇ mol of Mn++ per kg body weight to 100 ⁇ mol of Mn++ per kg body weight and 2 ⁇ mol of Ca++ per kg body weight to 1400 ⁇ mol of Ca++ per kg body weight.
• the method is applicable to the visualization of liver, kidney, pancreas, adrenal glands, heart, brain, salivary glands, gastrointestinal mucosa, uterus, tumors, the biliary system and the circulatory system.
• the source of Mn++ and the source of Ca++ may be administered simultaneously or separately, with Ca administration preceding Mn administration by up to 30 minutes.
• the method allows one to assess the metabolic activity of a tissue, distinguishing hyperactive or hypoactive states from normal.
• the method also allows one to assess tissue vascularity, blood flow and tissue perfusion.
• Figure 1 is a graph of normalized systolic blood pressure in which the change from baseline (in percent) is plotted as a function of time (in minutes);
• Figure 2 is also a graph of normalized systolic blood pressure in which the change from baseline (in percent) is plotted as a function of time (in minutes);
• Figure 3 is a graph of the ratio of MRI signal-to-noise as a function of time (in minutes) following administration of a composition of the invention.
• a Ca ion to Mn ion ratio of as high 1800: 1 could be considered without exceeding the Ca ion LD50; more conservatively, a ratio of 180: 1 could be contemplated by approaching l/10 th of the Ca ion LD50. If, however, the Mn ion dose is increased to lO ⁇ mol/kg, the ratio of Ca to Mn will not exceed 18: 1 if the conservative approach of remaining below l/10 th of the Ca ion LD50 is followed. In practice, it is not necessary to approach maximal amounts of added Ca to achieve a significant safety benefit.
• the particular ratio of Ca ion to Mn ion depends on the indication, the dose required to achieve efficacy and the desired safety margin (therapeutic index) for the Mn/Ca composition.
• improved utilization of the paramagnetic properties of manganese is achieved without the reduction in relaxivity or loss of agent through renal excretion associated with chelation.
• Manganese chelates demonstrate different phamacokinetics and pharmacodynamics for the manganese metal and the chelator components, suggesting dechelation. In such cases, one may see toxicity mediated through both the metal and the chelator.
• levels of Mn ion that would otherwise not be considered to be physiologically tolerable may be safely reached.
• compositions and methods of the invention are of utility in imaging a variety of metabolically active organs, in particular the liver, kidney, pancreas, heart, and adrenal glands.
• Manganese contrast enhancement is also useful for imaging of the gastrointestinal mucosa, uterus, salivary glands, brain, the biliary tree, and tumors.
• Mn ion binds to human serum albumin, alpha.2-macro globulin, transferrin, and other blood proteins with a concomitant increase in Mn relaxivity due to macromolecular association.
• This relaxivity increase coupled with the increased concentration of free manganese in the blood available for macromolecular association through the use of the compositions of this invention, provides an increase in signal intensity sufficient to allow even vascular imaging at biologically tolerable doses of Mn++.
• the Mn++/Ca++ compositions of the invention are approximately 20-fold more effective than the current gold standard gadolinium chelates in enhancing MR signal intensity in the blood, without suffering from contrast agent extravisation and rapid signal to noise degradation typical of extracellular fluid agents.
• compositions of the present invention may be used in place of manganese chelates to detect and assess myocardial ischemia and reperfusion using the technique described for the chelates in WO 99/01162.
• Mn is taken up by mitochondria, and Mn uptake correlates with dose across the clinically relevant range of dosing.
• Manganese uptake also correlates with blood flow to and metabolic activity of the relevant tissues.
• tissue uptake of Mn allows for the use of the compositions of the invention as viability markers (distinguish viable from non- viable tissue).
• the vascular enhancement due to Mn binding to plasma proteins also aids in defining blood flow and tissue perfusion (mL of blood flow per gram of tissue). Taken together the vascular and tissue phases of MR imaging with the Mn/Ca compositions of the invention allow for evaluation of the status of a variety tissues, organs and organ systems.
• compositions of the invention provide diagnostic utility for: (1) characterization of viable vs. non-viable tissue including the myocardium and tumors; (2) sizing of infarcts; (3) characterization of ischemic tissue or tissue at risk including the myocardium (reversible vs. irreversible damage); (4) assessment of tissue perfusion; and (5) characterization of vascular lesions proximal to areas of ischemia/infarct.
• PTCA interventional revascularization procedures
• stenting stenting
• by-pass interventional revascularization procedures
• one may predict the likelihood of success of interventional procedures (salvage of at risk tissue); one may evaluate and follow up treated patients (including for reperfusion); and one may assess the effect of therapy, including new drug therapy.
• compositions of the invention are also useful for the assessment of organ function before and after organ transplant, for example of heart, kidney and liver; for characterization of tumor (vascularity, metabolic activity, patterns of enhancement); for the assessment of liver disease and liver abnormalities, such as tumors (particularly to distinguish hepatocellular vs. non-hepatocellular tumor and benign lesions), cirrhosis and hemangioma; and for characterizing tissue vascularity (e.g., angiogenesis, tumor) and vascular lesions.
• tumor vascularity, metabolic activity, patterns of enhancement
• liver disease and liver abnormalities such as tumors (particularly to distinguish hepatocellular vs. non-hepatocellular tumor and benign lesions), cirrhosis and hemangioma
• tissue vascularity e.g., angiogenesis, tumor
• therapy including but not limited to drug, genetic, surgical, revascularization
• ischemic tissue, vascular lesions or tumor including but not limited to surgery, revascularization, transplantation, drug therapy, genetic therapy, new drug evaluation, radiation therapy, chemotherapy, tissue ablation therapy.
• compositions of the invention may be administered intravenously as a bolus or as an infusion over a period of time. Commonly, though not necessarily, the infusion will be over a period of 1 minute to 30 minutes.
• Mn gluconate /Ca gluconate (1 :8) is parenterally administered over periods ranging from 10 seconds to 20 minutes. Dosing is related to target organ of interest and may range from from 1 ⁇ mol/kg body weight to 100 ⁇ mol/kg body weight of a source of Mn++ ion together with from 2 ⁇ mol/kg body weight to 1400 ⁇ mol/kg body weight of a source of Ca++ ions.
• the source of manganese is administered at 2 ⁇ mol/kg body weight to 30 ⁇ mol/kg body weight and the source of calcium is administered at 4 ⁇ mol/kg body weight to 400 ⁇ mol/kg body weight.
• the source of manganese is administered at 3 ⁇ mol/kg body weight to 15 ⁇ mol/kg body weight and the source of calcium is administered at 6 ⁇ mol/kg body weight to 200 ⁇ mol/kg body weight.
• MRI is performed, according to methods well known to those skilled in the art, from during or immediately post dosing to 24 hours post dosing (vascular indications excepted).
• the rate of administration may be varied to further improve the cardiovascular tolerability of the contrast agent without an adverse effect on image quality, to increase the duration of the vascular phase of the agent, or to increase the dose without reducing the therapeutic index of the agent in order to enable imaging of target organs that accumulate manganese less efficiently than does liver.
• Advantages of the invention include: (1) Less of the administered dose is excreted renally (essentially none vs. 15 or so percent for MnDPDP); this effectively reduces the dose of Mn required for optimal image enhancement; (2) The solution relaxivity of manganese salts is maintained; (3) The risk of toxicity associated with the presence of chelator is avoided. For instance, loss of bioactive metals such as plasma Zn through chelation and renal excretion is avoided: (4) Rapid tissue enhancement is achieved relative to Mn chelates, allowing for earlier imaging; (5) Additional indications, for example imaging of tumors, as well as vascular and cardiac imaging are possible; and (6) The cost of the composition is lower than most chelate compositions and most compositions that employ rarer elements, such as gadolinium.
• the invention relates to a diagnostic composition
• a diagnostic composition comprising a source of a diagnostically effective quantity of Mn++ ion , a source of Ca++ ion and a pharmaceutically acceptable carrier for parenteral administration.
• diagnostically effective refers to an amount of Mn ion sufficient to increase the signal-to-noise ratio for MRI of the tissue in question. With present day instrumentation an increase of at least 5% is required to be diagnostically effective.
• source of Mn++ or Ca++ ion means any chemical species that can furnish a measurable concentration of Mn++ or Ca++ ion in normal saline or blood.
• the molar concentration of the salt when the source is a simple salt, soluble at the concentration employed, such as manganese chloride, manganese gluconate, or calcium gluconate, the molar concentration of the salt will be the molar concentration of the Mn++ or Ca++ ion.
• the source is a salt or complex that is not fully dissociated at the concentration employed, the molar concentration of Mn++ or Ca++ ions will be less than the molar concentration of the salt or complex, but its effective molarity can be readily calculated from the solubility product of the components by methods well known in the art, and the effective molarity of the metal ion can be experimentally determined with an ion-specific electrode, as well as similar methods well known in the art.
• concentrations of interest in the invention are those of the ions, not the concentrations of the source species, although the two are the same for simple, soluble salts.
• a soluble salt, for the purpose of the present invention refers to a salt which is essentially fully dissociated at the concentration being used.
• Antioxidants such as ascorbate, and stabilizers, such as calcium saccharate and borate complexes may be added to the compositions of the invention, as may other substances known in the pharmaceutical art to be useful in parenteral formulations.
• Formulations for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, pH modifiers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
• Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, water-for-injection (WFI) or the like, immediately prior to use.
• a sterile liquid carrier for example saline, water-for-injection (WFI) or the like
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders and granules according to methods well known in the pharmaceutical art. Sterile solutions of a unit dose for injection are preferred.
• a preferred composition for one embodiment of the invention is a sterile solution of 25 mM manganese salt and 200 mM calcium salt in WFI adjusted to pH 6.0 to 8.2 with sodium hydroxide and/or hydrochloric acid. About 4% to 8%, preferably 6%, by weight of the calcium is provided by calcium saccharate and the remainder by calcium glucon
• the source of manganese is preferably administered intravenously at 1 ⁇ mol/kg body weight to 100 ⁇ mol/kg body weight, more preferably at 2 ⁇ mol/kg body weight to 30 ⁇ mol/kg body weight. In many preferred embodiments, the source of manganese is administered intravenously at 3 ⁇ mol/kg body weight to 15 ⁇ mol/kg body weight.
• the source of calcium is preferably administered intravenously at 2 ⁇ mol/kg body weight to 1400 ⁇ mol/kg body weight, more preferably at 4 ⁇ mol/kg body weight to 400 ⁇ mol/kg body weight. In many preferred embodiments, the source of calcium is administered intravenously at 6 ⁇ mol/kg body weight to 200 ⁇ mol/kg body weight.
• tissue refers to such biological materials as gastrointestinal mucosa, tumor and the like.
• Organ refers to such biological organs as the liver, the kidney, the pancreas, the adrenal glands, the heart, the brain, salivary glands, and the uterus.
• System refers to such biological systems as the biliary system, the gastrointestinal system and the cardiovascular or circulatory system.
• the source of Mn++ and the source of Ca++ may be administered separately or in a single composition.
• the administration of a single parenteral dosage form will usually be simplest, but the clinician may employ any means known in the art to achieve the desired ratio of Mn++ to Ca++ in the individual being treated and to achieve the desired level of Mn++ in the tissue, organ or system of interest.
• administration of the two ions within 30 minutes of each other will generally be successful, but it is necessary to administer the calcium first, when sequential administration is employed.
• Example 1 Preparation of Manganese gluconate / Calcium gluconate 1 :X
• a 27.9mM Manganese gluconate stock solution was prepared as described below.
• the Mn gluconate used contained 5.5% water by assay.
• Ca gluconate and water for injection were added to the stock to prepare solutions of Mn gluconate and Ca gluconate of fixed molar ratio, as noted below. Without the addition of solubility enhancers, the concentration of the prepared solutions was limited to approximately 3% by weight Ca gluconate.
• the 27.9 mM Mn stock solution was prepared by adding 6.56g of Mn gluconate to 500mL of water for injection.
• the desired Mn Ca compositions can be made by adding Mn gluconate to commercially available Calcium Gluconate for Injection, 10% (232mM) which contains calcium saccharate as a solubility enhancing agent.
• Mn/Ca 1 8 [29 mM in Mn, 232 mM in Ca(II)]
• Ten milliliters of Calcium Gluconate Injection, 10% is added to 136 mg of Mn gluconate.
• Contrast media of the invention with molar Ca to Mn ratios of 1 : 1 to 8: 1, prepared according to the description in Example 1, were studied for their effects on systolic blood pressure following intravenous administration in New Zealand White Rabbits.
• the Mn++ dose for all compositions was held constant at about 30 ⁇ mol/kg, a dose previously shown to induce a minimum 20% drop in systolic blood pressure. Changes in blood pressure were monitored via an in-dwelling femoral artery catheter. Blood pressure baselines were re-established between doses.
• Table 1 summarizes the findings in a study in which the Ca/Mn ratio was doubled with each successive dose.
• Table 2 summarizes findings in a second experiment focused on the more effective range of ratios studied in the exploratory experiment.
• Figure 1 illustrates the change in normalized systolic blood pressure over time for Mn and for several Mn /Ca compositions of different molar ratios, all administered intravenously in the rabbit at a constant Mn++ ion dose of about 30 ⁇ mol/kg.
• Table 3 shows data collected in a naive rabbit for Mn dosed at about 30 ⁇ mol/kg and Ca at 240 ⁇ mol/kg, individually and as a mixture, following the same protocol used in Example 2.
• Example 4 Mn/Ca compositions in the rat
• a 1 : 8 mole ratio Mn / Ca contrast agent of this invention prepared according to the description in Example 1 , was studied for effects on systolic and mean blood pressure relative to a Mn gluconate control in Wistar rats. Agents were administered intravenously at a Mn dose of lOO ⁇ mol /kg. Changes in blood pressure were monitored via an in-dwelling femoral artery catheter. The results, shown in Table 4, confirm that in the rat, as well as the rabbit, the combination of Mn and Ca produces less depression of hemodynamic parameters than does Mn alone, thus permitting the safe administration of a diagnostically effective dose.
• the blood phamacokinetics of Mn / Ca (1 : 8) were examined by MRI in the rabbit.
• the contrast agent was administered intravenously in the ear vein over 1 minute at a dose of 10 ⁇ mol/kg Mn.
• Blood samples (3mL each in heparinized tubes) were taken from the ear artery prior to and immediately, 2, 10, 20, 30 and 60 minutes post administration of contrast.
• the signal intensity data shown in Table 7, demonstrate pharmacokinetics consistent with those found for Mn++, when measured with a 56 Mn tracer. (Borg, D.C.; Cotzias, G.C. J Clin Invest. 37: 1269)
• Tl shortening seen with Mn/Ca (1 :8) in saline was compared to that seen in blood.
• Tubes of saline and blood (3mL each) were spiked with 100%), 50% and 10%) of the intravascular concentration of Mn / Ca (1 :8) that would be expected if it were administered as a bolus at a dose of lO ⁇ mol/kg Mn.
• Gd DTPA was spiked in 3mL of saline and blood at 100% of the expected intravascular concentration following bolus dosing at lOO ⁇ mol/kg, a dose typically used in the clinic for vascular imaging.
• MnDPDP which does not bind to plasma proteins, has a solution Rl of 1.90 s _1 mM _1 at 40 °C and 20MHz.
• GdDTPA has an Rl of 3.84 s " 'mM " ' under similar conditions.

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