Abstract:
A contrast agent adapted for magnetic resonance imaging of a sample, the contrast agent comprising a suspension in a medium acceptable for magnetic resonance imaging of (a) coated particles of a contrast agent possessing paramagnetic characteristics and (b) coated particles of a contrast agent possessing diamagnetic characteristics, each of the coatings being selected from a group of materials which [I] renders the coated particles (a) and (b) substantially comparable with and substantially biologically and substantially chemically inert to each other and the environments to which the contrast agent is exposed during magnetic resonance imaging and [II] which substantially stabilizes the suspension; the nature of each of the coatings and the relative amounts of (a) and (b) in the suspension being such that the positive magnetic susceptibility of (a) substantially offsets the negative magnetic susceptibility of (b) and the resulting suspension has substantially zero magnetic susceptibility and, when employed in magnetic resonance imaging, results in the substantial elimination of imaging artifacts. Also disclosed is a method of imaging a sample which comprises introducing into the sample the above-described contrast agent and generating an image thereof.

Description:
RELATED APPLICATION 
     This is a continuation-in-part of application Ser. No. 07/925,549 filed Aug. 7, 1992, now U.S. Pat. No. 5,323,780. 
     Research leading to the completion of the invention was supported, in part, by IRB Grant No. 70-91 provided by Advanced Magnetics, Inc. of Cambridge, Mass., and IRB Grant No. 457-90 provided by E-Z-EM, Inc. of Westbury, N.Y. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to imaging contrast agents, in particular, NMR imaging contrast agents. 
     2. Discussion of the Prior Art 
     It is well known to enhance NMR (nuclear magnetic resonance) images by, prior to conducting the imaging, introducing into the sample to be imaged ferromagnetic, diamagnetic or paramagnetic particles which shadow the image produced to intensify and contrast the image generate by the NMR sensitive nuclei. See, for example, the disclosures of U.S. Pat. Nos. 4,731,239; 4,863,715; 4,749,560; 5,069,216; 5,055,288; 5,023,072; 4,951,675; 4,827,945 and 4,770,183, the entire contents and disclosures of all of which are incorporated herein by reference. 
     A disadvantage associated with the use of NMR image contrast agents, however, resides in the fact that the internal magnetic properties of the various contrast agents employed heretofore affect the applied external field during the imaging process. These interactions of the paramagnetic or diamagnetic properties of the contrast agents with the applied external magnetic field give rise to irregularities in the resulting image, commonly referred to as imaging artifacts. 
     The application of magnetic resonance imaging (MRI) to abdominal imaging is potentially revolutionary, but currently is seriously limited by the inability to distinguish tumors, abscesses and other abnormalities from normal structures, e.g., bowel. As for computed tomography, the use of oral or rectal contrast agents is expected to dramatically improve the diagnostic value of MRI for abdominal diseases. Currently, superparamagnetic iron oxide (SPIO) is the best oral contrast agent available for use in MRI. 
     Iron oxide acts as a gastrointestinal (GI) MRI contrast agent primarily due to the dipolar interaction of the unpaired electrons of iron with the water protons in the body. In addition, its internal magnetization aligns parallel to an applied external field, increasing the magnetic field strength nearby. This is known as positive magnetic susceptibility, and also leads to large field gradients near the articles. Both of these mechanisms cause loss of signal intensity within the bowel. This loss of signal causes the desired enhancement of the difference in the appearance of bowel and other normal and abnormal structures in magnetic resonance images. 
     A problem with SPIO suspensions in the past has been that they not only affect the volume that actually contains the suspension (i.e., bowel), but also lead to artifactual signal loss in the region surrounding this area. This phenomenon is called susceptibility artifact and is more pronounced with greater field strength and with gradient recalled echo pulse sequences. This artifact reduces the diagnostic usefulness of SPIO, especially for gradient echo sequences which otherwise have many advantages for MRI of the abdomen. 
     Barium sulfate suspensions are also used as oral contrast agents for GI MRI. Barium sulfate is a diamagnetic substance; its paired electrons interact with an external magnetic field to produce internal magnetization oriented opposite to the applied field. This is known as negative magnetic susceptibility, and also produces loss of signal intensity due to diffusion of water molecules through the field gradients around the particles. 
     Imaging contrast agents which do not suffer from the above-noted disadvantages and a method of imaging which produces a substantially artifact-free image are described in U.S. Pat. No. 5,323,780 of which the present application is a continuation-in-part. Briefly, the improvements described in U.S. Pat. No. 5,323,780 related to a contrast agent useful for imaging a sample comprising a suspension in a medium acceptable for imaging (a) particles of a contrast agent possessing paramagnetic characteristics and (b) particles of a contrast agent possessing diamagnetic characteristics and a method of imaging a sample which comprises introducing into the sample the above-described contrast agent and generating a substantially artifact-free image thereof. 
     It has been found that the contrast agent suspensions described in U.S. Pat. No. 5,323,780 are subject to instability in that the components thereof tend to interact with each other and with the environments in which they are employed for magnetic resonance imaging such that the suspension of particles (a) and (b) destabilize and the particles &#34;settle out&#34; of suspension. 
     It is an object of the present invention to provide novel imaging contrast agents which do not suffer from the disadvantages associated with the agents described in U.S. Pat. No. 5,323,780, as well as a novel method of imaging which produces a substantially artifact-free image. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a graphical depiction comparing the relaxivities of contrast agents comprising (a) mixture of ferrite and barium sulfate and (b) ferrite alone. 
     FIG. 2 depicts the magnetic susceptibility artifacts produced by various suspensions of barium sulfate and ferrite. 
     FIGS. 3a-3e show proton MR images of the contrast agent suspensions: 
     (a) T 1  -weighted image, TR=300 ms, TE=20 ms 
     (b) &#34;proton density&#34; image, TR=1800 ms, TE=30 ms 
     (c) T 2  -weighted image, TR=1800 ms, TE=90 ms 
     (d) gradient-recalled echo image, TR=100 ms, TE=40 ms, 30° flip angle 
     (e) gradient-recalled echo image, TR=100 ms, TE=40 ms, 30° flip angle. 
     Sample suspensions in 2.5% w/v aqueous vehicle are, top to bottom in FIGS. 3a-3d: 
     (1) Fe100: Fe 3  O 4 , 175 mg Fe/liter 
     (2) Fe75/Ba25: Fe 3  O 4 , 131.25 mg Fe/liter, and BaSO 4 , 150% w/v 
     (3) Fe50/Ba50: Fe 3  O 4 , 87.5 mg Fe/liter, and BaSO 4 , 100% w/v 
     (4) Fe25/Ba75: Fe 3  O 4 , 43.75 mg Fe/liter, and BaSO 4 , 50% w/v 
     (5) Ba100: BaSO 4 , 200% w/v. 
     For FIG. 3e, sample suspensions in 2.5% w/v aqueous vehicle are, top to bottom: 
     (1) Fe100: Fe 3  O 4 , 175 mg Fe/liter 
     (2) Fe25: Fe 3  O 4 , 43.75 mg Fe/liter 
     (3) Fe25/Ba75: Fe 3  O 4 , 43.75 mg Fe/liter, and BaSO 4 , 50% w/v 
     (4) Ba100: BaSO 4 , 200% w/v. 
     FIG. 4 shows transverse relaxation rates R 2  as a function of the echo spacing TE/n (=2τ) for the Fe100 and Fe25/Ba75 suspensions, as well as the sum of the rates for the Fe25 and Ba75 suspensions. Points represent the mean of 3-5 individual determinations. To preserve clarity of presentation, error bars are not shown; the mean standard deviation expressed as percent of the mean is 14.5%±5.7%. 
     SUMMARY OF THE INVENTION 
     The foregoing and other objects are realized by the present invention, one embodiment of which relates to a contrast agent adapted for magnetic resonance imaging of a sample, the contrast agent comprising a suspension in a medium acceptable for magnetic resonance imaging of (a) coated particles of a contrast agent possessing paramagnetic characteristics and (b) coated particles of a contrast agent possessing diamagnetic characteristics, each of the coatings being selected from a group of materials which [I] renders the coated particles (a) and (b) substantially compatible with and substantially biologically and substantially chemically inert to each other and the environments to which the contrast agent is exposed during magnetic resonance imaging and [II] which substantially stabilizes the suspension; the nature of each of the coatings and the relative amounts of (a) and (b) in the suspension being such that the positive magnetic susceptibility of (a) substantially offsets the negative magnetic susceptibility of (b) and the resulting suspension has substantially zero magnetic susceptibility and, when employed in magnetic resonance imaging, results in the substantial elimination of imaging artifacts. 
     A further embodiment of the invention is directed to a method of imaging a sample which comprises introducing into the sample the above-described contrast agent and generating an image thereof. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     It will be understood by those skilled in the art that for purposes of describing the present invention, the terms discussed hereinbelow have the following meanings and definitions. 
     The term &#34;paramagnetic&#34; as used herein is intended to include those substances described as &#34;ferromagnetic&#34; and &#34;superparamagnetic&#34; as well as paramagnetic, all of which have positive magnetic susceptibility characteristics. 
     The phrase &#34;. . . the positive magnetic susceptibility . . . offsets the negative magnetic susceptibility . . . to produce . . . a substantially zero magnetic susceptibility . . . &#34; is intended to mean the respective positive and negative magnetic susceptibilities are matched so as to produce a contrast agent which results in an artifact-free image. This &#34;matching&#34; may not necessarily produce a zero magnetic susceptibility since the suspending medium and any other components present in the suspension may have an inherent magnetic susceptibility which must be taken into account when balancing the amounts of paramagnetic and diamagnetic particles to produce a contrast agent which yields an artifact-free image upon imaging. 
     The term &#34;substantially stable suspension&#34; as used herein refers to suspensions of particles (a) and (b) which are stable against settling out, i.e., remains fluid, for a significant period of time, i.e., for at least about 3-4 hours. 
     The term &#34;coating material&#34; refers to any material capable of being coated on particles (a) and (b) which is compatible and biologically and chemically inert with respect to the particles themselves, each other and the environments to which the contrast agent is exposed during the imaging process. 
     The coating may be applied to the particles according to any conventional particle coating technique, with which those skilled in the art are well aware. 
     The thickness of the coating on the particles is not overly critical. Care should be taken to ensure that the coated particles maintain their magnetic characteristics and that the suspensions thereof remain stable. Generally, a coating thickness of from about 0.1 to about 2 microns is preferred. 
     Any suitable diamagnetic material possessing negative magnetic susceptibility characteristics may be employed in the practice of the invention. Barium sulfate, preferably in the form of a mixture of barites and substantially pure barium sulfate, is exemplary of suitable diamagnetic substances. 
     Any suitable paramagnetic material, e.g., iron oxide (ferrite), possessing positive magnetic susceptibility characteristics may be employed in the practice of the invention. Particularly preferred is superparamagnetic iron oxide (SPIO). 
     Any suitable suspending medium may be employed for preparing suspensions of the respective paramagnetic and diamagnetic particles, as well as the mixture thereof. For example, an aqueous solution of vegetable gums (e.g., 2.35% w/v) may be employed to prepare a suspension of barium sulfate. Water may be used to prepare the suspension of SPIO. Those skilled in the art are well aware of suitable media for use in preparing image contrasting agents. It is preferred, of course, when the agent is to be employed in NMR imaging of a patient (human or non-human) that the medium comprise a physiologically acceptable one. 
     Amounts of the respective paramagnetic and diamagnetic particles are employed so as to produce a mixture, the use of which as a contrasting agent yields an artifact-free image. 
     An example of such a suspension is one comprising a mixture of (1) a 25% v/v suspension of SPIO in water (175 mg/liter) and (2) a 75% v/v suspension of a mixture of 56.8% by weight of Barytes #1 and 43.2% by weight of USP BaSO 4  in a 2.35%w/v aqueous solution (220%w/v=80%w/w). This suspension, when employed as an imaging contrasting agent in an NMR imaging method, produces good contrast in all pulse sequences with no artifactual images. 
     The combination of materials with positive and negative magnetic susceptibilities in a suspension results in increased field gradients between suspended particles with improved relaxivity on a microscopic scale. On a macroscopic scale, mixing the two suspensions (one with positive and one with negative magnetic susceptibility) in the correct proportions produces a suspension with zero bulk susceptibility and no artifacts. 
     It has been found that a combination of 25% v/v SPIO (175 mg/liter) and 75% v/v barium sulfate suspension (220% w/v) shows no artifact and produces good contrast in all pulse sequences. FIG. 1 graphically demonstrates the improved relaxivity of a 50% v/v AMI 121/50% v/v Liquid HD suspension over the sum of the relaxivities of each of its components. FIG. 2 demonstrates the magnetic susceptibility artifacts produced by different suspensions of barium sulfate and ferrite in a gradient echo pulse sequence. Note that suspension 2 does not produce any artifact. In FIG. 2, 
     1=pure Liquid HD 
     2=25% SPIO/75% Liquid HD 
     3=50% SPIO/50% Liquid HD 
     4=75% SPIO/25% Liquid HD 
     5=pure SPIO suspension 
     6=50% SPIO/50% water 
     7=25% SPIO/75% water 
     8=pure water. 
     The coating material may be any substance capable of forming a coating on the particles (a) and (b) which is inert and compatible as described above. 
     Suitable coating materials include natural and synthetic polymers, gums or resins, polysaccharides, proteinaceous materials, cellulosic materials and silicones. 
     Exemplary of natural polymer coating materials are rubber, celluloses, gums and resins. 
     Suitable specific coating materials include guar gum or its derivatives such as hydroxypropyl-, carboxymethyl- and carboxymethylhydroxypropyl-guar; xanthan gum or its derivatives; gum arabic or its derivatives; tragacanth gum or its derivatives; dextran, mannan, xylan, fructan, arabinan, pectin; methyl-, carboxymethyl-, ethyl-, hydroxyethyl-, hydroxypropyl-cellulose; albumin, gamma globulin, heparin; polydimethyl-, polydibenzylsiloxane; polystyrene, polyethylene, polypropylene, polymethylmethacrylate, polyurethane, polyacrylic, polyamide 6/6 (nylon) and polystyrene/glycidyl methacrylate. 
     EXAMPLE 1 
     Powdered Fe 3  O 4  (a Fe(III)/Fe(II) mixture) was obtained from Alfa/Johnson Matthey. The Fe 3  O 4  particle size was 0.3-0.4 μm. The vehicle used for particle suspension and BaSO 4  (particle size 3 μm) were obtained from E-Z-EM, Inc. The BaSO 4  and Fe 3  O 4  were mixed dry as powders with the vehicle and then diluted with water [Tilcock et al, J. Magn. Reson. Imaging, Vol. 1, page 463 (1991)]. Final stock concentrations were 200% BaSO 4  (Ba100), 175 mg Fe/liter Fe 3  O 4  (Fe100), and 2.5% w/v vehicle. The Fe 3  O 4  suspension was mixed with the BaSO 4  suspension in 1:3 (Fe25/Ba75), 1:1 (Fe50/Ba50), and 3:1 (Fe75/Ba25) volume ratios; a 1:3 dilution of the Fe 3  O 4  suspension (Fe25) and a 3:1 dilution of the BaSO 4  suspension (Ba75) were made with 2.5% w/v vehicle solution to yield a 43.75 mg Fe/liter Fe 3  O 4  suspension and a 150% w/v BaSO 4  suspension. The samples (20 mL) were placed in 2.5 cm diameter glass bottles in a water bath phantom. 
     Images were obtained at 1.5 T (GE Signa) with a quadrature head coil using T 1  -weighted (TR=300 ms, TE=20 ms, 1 NEX, 256×128 matrix, 28 cm FOV, 5 mm slice) and T 2  -weighted (TR=1800 ms, TE=30/90 ms, 1 NEX, 256×128 matrix, 28 cm FOV, 5 mm slice) spin echo and GRASS gradient echo (TR=100 ms, TE=30, 40, 60 ms; TR=80 ms, TE=40 ms; TR=50 ms, TE=25 ms; 30° flip angle; 4 NEX; 256×128 matrix; 28 cm FOV; 5 mm slice) sequences. 
     For measurement of T 1  and T 2  relaxation times, a 2T SISCO (Spectroscopy Imaging Systems Corp.) system was used. Calculations were performed with the VNMR software on a Sun SPARC 2/GX workstation. The inversion recovery pulse sequence with TR=10-15 s (≧5T 1 ) and TI ranging from 0.01 s to 13 s (10-12 values per measurement) was used for T 1  determination. A multiple echo Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with TR=6-13 s (≧5T 1 ) and 2τ=1, 1.5, 2, 2.5, 3, 4, 6, 10, 15, 25, 35 ms (TE=2τn, where n is the number of echoes and 2τ is the echo spacing) was used for T 2  measurements. The number of echoes was varied for each value of the echo spacing 2τ (=TE/n) of the CPMG sequence to generate the transverse magnetization decay curve from which T 2  was calculated. In order to characterize the effect of diffusion on the signal decay, the relaxation rate (=1/T 2 ) as a function of 2τ, R 2  (2τ), was plotted versus TE/n (=2τ). 
     FIG. 3 shows images of the phantoms obtained at 1.5 Tesla with different imaging sequences. FIG. 3a is a T 1  -weighted image (TR=300 ms, TE=20 ms), FIG. 3b is a &#34;proton density&#34; image (TR=1800 ms, TE=30 ms), FIG. 3c is a T 2  -weighted image (TR=1800 ms, TE=90 ms), and FIG. 3d is a gradient-recalled echo image (TR=100 ms, TE=40 ms, 30° flip angle). Suspensions containing Fe 3  O 4  appeared very dark and suspensions containing BaSO 4  appeared dark on &#34;proton density&#34; and T 1  - and T 2  -weighted images. All suspensions appeared dark in gradient echo images. These results are indicative of short T 2  for BaSO 4  and very short T 2  for Fe 3  O 4 . On gradient echo images, concentrated BaSO 4  suspensions produced some artifacts and Fe 3  O 4  suspensions produced extensive artifacts. The 1:3 Fe 3  O 4  :BaSO 4  (Fe25/Ba75) mixture, however, produced no artifact. This was not due to dilution effects, as individual suspensions of Fe 3  O 4  (Fe25, FIG. 3e) and BaSO 4  (Ba75) at the concentrations found in the 1:3 mixture did produce artifacts. 
     To better characterize the relaxation properties of the suspensions, T 1  and T 2  measurements were made at 2.0 Tesla. Table 1 shows the results of the T 1  experiments. The T 1  of the Fe100 sample is 1.17 s, those of the Fe25 and Ba75 samples are about twice as long, and that of the Fe25/Ba75 sample is intermediate between that of the Fe100 sample and those of the Fe25 and Ba75 samples. The T 1  obtained by summing the rates of the Fe25 and Ba75 samples is the same as that of the Fe100 sample. FIG. 4 shows the transverse relaxation rates R 2  as a function of the echo spacing TE/n (=2τ) for the Fe100 and Fe25/Ba75 suspensions, as well as the sum of the rates of the Fe25 and Ba75 suspensions. The R 2  values for the Fe100 sample are slightly, but not significantly, greater than those of the Fe25/Ba75 sample. The R 2  values for the Fe100 and Fe25/Ba75 samples are greater than those obtained by summing the rates of the Fe25 and Ba75 suspensions, except for short echo times (TE/n&lt;2 ms), where all the rates converge to similar values. All the suspensions have a significant contribution to R 2  from diffusion of water through field gradients around the suspended particles, as exhibited by the increase in R 2  with increasing echo spacing. 
     Water is diamagnetic with a negative susceptibility (X=-13×10 -6  cgs units). Barium sulfate is even more diamagnetic (X=-71.3×10 -6  cgs units). Iron oxide is paramagnetic with a strongly positive magnetic susceptibility. If suspensions of particles with opposing susceptibilities are mixed in the proper proportions, the bulk susceptibility of the resulting suspension can be made to match that of water. In this case, there will be no susceptibility artifact in a gradient echo image (FIG. 3). However, the favorable relaxation properties which make these suspensions good negative contrast agents are retained (FIG. 4). 
     The combination of diamagnetic BaSO 4  and SPIO in proper proportions results in a susceptibility-matched suspension which produces no artifacts in gradient echo images, yet retains a short T 2  which is useful in producing negative contrast. This mixture is useful as an improved oral negative contrast agent for use in gastrointestinal MRI. 
     
                       TABLE 1______________________________________Spin-Lattice Relaxation Times (T.sub.1)and Rates (R.sub.1) Measured at 2.0 TeslaSample.sup.a     T.sub.1 (s).sup.b                      R.sub.1 (1/s).sup.b______________________________________Fe100            1.17 ± 0.04                      0.855 ± 0.033Ba75             2.43 ± 0.22                      0.413 ± 0.036Fe25             2.32 ± 0.17                      0.432 ± 0.032Sum (Ba75 + Fe25)            1.18 ± 0.10                      0.845 ± 0.068Fe25/Ba75        1.60 ± 0.08                      0.626 ± 0.032______________________________________ .sup.a All samples contain 2.5% w/v vehicle to keep particles in suspension. .sup.b Values are means ± S.D. 
    
     EXAMPLE 2 
     A dry powder of 0.3 gm of the solid particles (either Fe 3  O 4  or BaSO 4 , 0.1-10 micron diameter) is uniformly suspended, by homogenization and/or sonication, in a 15% (w/v) solution of polystyrene in toluene. The suspension is then slowly added, with homogenization and/or sonication, to 100 mL of a 5% (w/w) aqueous polyvinylalcohol solution, followed by addition of 100-150 mL of methanol. The resulting suspension of polystyrene-coated particles is then dispersed and diluted into about 750 mL of water and centrifuged, and the supernatant is discarded. The coated particles are thoroughly dried and then suspended in a 1.25% aqueous solution of carboxymethylcellulose. The T 2  relaxation time of a 0.07% (w/v) suspension of coated Fe 3  O 4 , measured at 4.7 Tesla, is about 17 msec, which compares favorably with the value measured for uncoated Fe 3  O 4 . 
     EXAMPLE 3 
     The imaging procedure of Example 1 was employed utilizing suspensions prepared according to Example 2 with similar results. The suspensions prepared according to Example 2 remained stable throughout the procedure.