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Description  |
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FIELD OF INVENTION
The field of this invention relates to magnetic resonance (MR) imaging
(MRI) of the human body and to the use of paramagnetic contrast agents to
improve the diagnostic usefulness of the MR images. More particularly,
this invention is concerned with a method of MRI examination of the liver
and spleen.
BACKGROUND OF THE INVENTION
MR imaging of the human body can display both normal anatomy and a variety
of organ pathologies, including tumors. For example, the liver, pancrease,
spleen and gall bladder can be imaged by tomographic slides in various
planes. The techniques used for MRI liver examination have included
delineation by spin-echo, inversion recovery, and saturation recovery
pulse sequences but definition of normal from abnormal has not been
predictable. In hepatic MRI, specifically, contrast resolution of the
hepatic images varies greatly depending on the data acquisition technique
employed to obtain the image, although tumors associated with the liver or
spleen usually result in prolongation of both the longitudinal (T.sub.l)
and the transverse (T.sub. 2) relaxation times as compared with normal
tissues. Earlier reports have emphasized the importance of using
paramagnetic agents to increase the T.sub.l differences between normal and
pathologic tissues and considered coincidental T.sub.2 diminution an
impediment.
Paramagnetic contrast agents as free metal ions, chelates, or insoluble
metal compounds have been described for use in enhancing intrinsic
contrast in MR imaging. Such paramagnetic metals include gadolinium,
chromium, copper, manganese, and iron. Because of possible toxicity,
soluble chelates have been suggested for parenteral administration and
insolubilized compounds for oral administration. Heretofore, however, the
targeting of stable contrast agents to the liver and spleen has not been
satisfactory.
An effective, safe reticuloendothelial system (RES) MRI contrast agent
which can increase the sensitivity and differentiation of normal and
pathologic tissue in the liver or spleen has not been previously
described. This problem is aggravated by the fact that existing modalities
for other imaging procedures for the liver and spleen have approximately a
10-20% false-negative rate for detecting hepatic metastases, and a 40-50%
false-negative rate for detecting lymphomatous involvement, necessitating
laproscopic staging. Further, as pointed out above, tumor involvement of
liver, spleen and other tissues has consistently been shown to increase
T.sub.1 and T.sub.2 relaxation parameters to a variable and unpredictable
degree, which also results in a high incidence of false negatives. RES
agents are useful because liver replaced by tumor does not possess RES
cells and therefore does not take up the contrast agent. Non-RES agents
more randomly distribute between normal and pathologic tissue.
Hepatic disease conditions resulting in abnormally high levels of iron in
the liver have been shown to produce alterations of tissue relaxation
times as observed by MRI. See, for example, Doyle, et al., Am. J.
Roentgenol. (1982) 138: 193-200; Stark, et al., Radiology (1983) 148: 743
-751; and Runge, et al., Am. J. Roentgenol. (1983) 141: 943 -948. Observed
decreases in T.sub.l have been attributed either to paramagnetic
enhancement of longitudinal relaxation, or to alterations of hydrated
tissue proteins. Heretofore, the production of T.sub.2 dimunition as seen
in these disease states has not been produced with a potent, safe contrast
agent. Soluble iron compounds have been tested as MRI contrast agents.
Wesbey, et al., Radiology (1983) 149: 175 -180.
SUMMARY OF INVENTION
The method of the present invention for MRI examination of the liver and/or
spleen utilizes encapsulated particulate superparamagnetic contrast agents
in the form of sized microspheres. Preferably, the contrast agent is a
ferromagnetic iron compound in a particle size of not over 300 Angstroms.
By utilizing microspheres within of sizes up to 8 microns, such as 2 to 5
microns, the parenterally administered contrast agents are rapidly
segregated by the reticuloendothelial system and concentrated in the liver
and spleen. Effective segregation and concentration in these organs can
occur in as short a time as 1 to 10 minutes. Only a small quantity of the
microspheres needs to be administered for effective reduction of the
T.sub.2 relaxation time of the subject's liver and/or spleen. The method
emphasizes T.sub.2 differences between normal and pathological tissues,
improving visual contrast without losing anatomical detail. Because of the
effectiveness of the targeting to the liver and spleen, toxicity reactions
and other side effects are minimized. A preferred form of ferromagnetic
iron is the magnetic oxide of iron known as magnetite.
The advantages of the encapsulated contrast agents of the present invention
for hepatic and splenic imaging include the following: (1) superior
targeting as compared with contrast agents that are distributed primarily
in the blood pool and interstitium, or metabolized by hepatocytes; (2) the
contrast material may be composed of physiologic concentrations of normal
physiologic substrate, such as human serum albumin matrix and iron oxides,
which can be metabolized, for example, by normal physiologic mechanisms of
clearance, (3) toxicity can be minimized by rapid clearance by
reticuloendothelial system when administered intravenously which protects
the metabolically functional hepatocytes from exposure to the contrast
material (viz. the first pass clearance can be approximately 80%); (4)
rapid intravenous clearance by the target liver and spleen is predictable
with a normal functioning reticuloendothelial system; and (5) the
microspheres can be nonembolic in size and need not be trapped by the
proximal arteriolar bed. By employing the method of this invention to
reduce the transverse relaxation time (T.sub.2) in MR imaging of the liver
and spleen, tumors may be observed with much greater accuracy and clarity.
The concentration in the liver and spleen of the encapsulated contrast
agent by "blackening" the normal tissue highlights on areas replaced by
tumor.
Particulate ferromagnetic compounds of not over 300 Angstroms in size
exhibit superparamagnetic properties. They provide a much larger magnetic
susceptibility than ordinary paramagnetic materials. Further,
magnetization increases with increases applied external field, but the
magnetization is rapidly lost when the superparamagnetic particles are no
longer subjected to an applied field. The low residual magnetization
characteristic of such superparamagnetic particles avoids clumping or
aggregation of the microspheres which might otherwise occur. MR equipment
generates magnetic fields which could otherwise result in more permanent
magnetization of the microspheres. By minimizing clumping of the
microspheres more effective and uniform biodistribution can be obtained.
DETAILED DESCRIPTION
The superparamagnetic contrast agent is used in particulate form, for
example, as particles of 50 to 300 Angstroms diameter. Particle size of
not over 300 Angstroms provides ferromagnetic iron compounds with the
desired superparamagnetic characteristics; namely, enhanced magnetic
susceptibility and low residual magnetization. Preferably, the particulate
forms are substantially water-insoluble, such as insoluble oxides or
salts. The superparamagnetic contrast agent may also be in the form of
particles of an elemental metal such as particularly iron particles sized
below 300 Angstroms.
A preferred particulate contrast agent is magnetite, which is a magnetic
iron oxide sometimes represented as Fe.sub.3 O.sub.4 (or as FeO.Fe.sub.2
O.sub.3 .) Commercially, fine powders or suspensions of magnetite are
available from Ferrofluidics Corporation, Burlington, Massachusetts. The
size range of the particles is submicron, viz. 50 to 200 Angstroms. Other
water-insoluble superparamagnetic iron compounds can be used such as
ferrous oxide (Fe.sub.2 O.sub.3), iron sulfide, iron carbonate, etc.
For purposes of this invention, the microspheres comprise relatively
spherical particles consisting of protein, carbohydrate or lipid as the
biodegradable matrix for the paramagnetic contrast agent. For effective
targeting to the liver and spleen, the microspheres comprising the
encapsulated contrast agents should have diameters up to about a maximum
size of 8 microns. An advantageous size range appears to be from about 2
to 5 micro diameter. Less than 1.5 micron microspheres can be used as a
livery spleen contrast agent (viz. 1.0 micron size), but circulation time
is prolonged, that is, fewer spheres will be rapidly taken up by the RES.
Microspheres of larger size than 8 microns may be sequestered in the first
capillar bed encountered, and thereby prevented from reaching the liver
and spleen at all. Large microspheres (viz. 10 microns or more) can be
easily trapped in the lungs by arteriolar and capillary blockade. See
Wagner et al., J. Clin. Investigation (1963), 42:427; and Taplin, et al.,
J. Nucl. Medicine (1964) 5:259.
The matrix material may be a biodegradable protein, polysaccharide, or
lipid. Non-antigenic proteins are preferred such as, for example, human
serum albumin. Other amino acid polymers can be used such as hemoglobin,
or synthetic amino acid polymers including poly-L-lysine, and
poly-L-glutamic acid. Carbohydrates such as starch and substituted (DEAE
and sulfate) dextrans can be used. (See Methods in Enzymology, 1985, Vol.
112, pages 119-128). Lipids useful in this invention include lecithin,
cholesterol, and various charged phospholipids (stearyl amines or
phosphatidic acid). Microspheres having a lipid matrix are described in
U.S. Pat. No. 4,331,564.
Microspheres for use in practicing the method of this invention can be
prepared from albumin, hemoglobin, or other similar amino acid polymers by
procedures heretofore described in literature and patent references. See,
for example, Kramer, J. Pharm. Sci. (1974) 63: 646; Widder, et al., J.
Pharm. Sci. (1979) 68: 79; Widder and Senyei, U.S. Pat. No. 4,247,406; and
Senyei and Widder, U.S. Pat. No. 4,230,685.
Briefly, an aqueous solution is prepared of the protein matrix material and
the paramagnetic/ferromagnetic contrast agent, and the aqueous mixture is
emulsified with a vegetable oil, being dispersed droplets in the desired
microsphere size range. Emulsification can be carried out at a low
temperature, such as a temperature in the range of 20-30.degree. C., and
the emulsion is then added dropwise to a heated body of the same oil. The
temperature of the oil may range from 70 to 160.degree. C. The dispersed
droplets in the heated oil are hardened and stabilized to provide the
microspheres which are then recovered. When most of the microspheres as
prepared, such as 80% or more, have sizes within the ranges described
above, they can be used as prepared. However, where substantial amounts of
oversized or undersized microspheres are present, such as over 10 to 20%
mof microspheres larger than 8 microns, or over 10 to 20% of microspheres
smaller than 1.5 microns, a size separation may be desirable. By the use
of a series of micropore filters of selective sizes, the oversized and
undersized microspheres can be separated and the microspheres of the
desired size range obtained.
The microspheres may contain from 5 to 100 parts by weight of the contrast
agent per 100 parts of the matrix material. For example, in preferred
embodiments, microspheres can contain from 10 to 30 parts by weight of
magnetite particles or another superparamagnetic contrast agent per 100
parts of matrix material such as serum albumin.
Intravenous parenteral infusion is the preferred administration route for
the microspheres. However, selective intra-arterial injection/infusion can
be employed. Where the microspheres contain from 10-40 parts by weight of
the contrast agent per 100 parts of the matrix material does within the
range from 1 to 40 milligrams per kilogram of body weight of the human
subject can be used. For example, typical doses are 5 to 15 mg/kg. With
albumin microspheres containing 20% magnetite, the amount administered may
comprise 10 mg of the microspheres per kg body weight. For administration,
the microspheres may be suspended in a sterile solution of normal saline.
In practicing the method of the inventions, the microspheres containing the
contrast agent are parenterally administered prior to the MRI examination.
The examination is delayed until the microspheres have been segregated by
the reticuloendothelial system and are concentrated in the liver and
spleen. A suitable period of delay is from about 1 to 10 minutes. The MRI
examination is then carried out in the usual manner to obtain images of
the liver and/or spleen. The agent is efficaceous for T.sub.2, T.sub.2 and
mixed T.sub.2 and T.sub.2 weighted pulse sequences. The T.sub.2 and mixed
sequences are preferred.
EXPERIMENTAL
The method of this invention was tested on an experimental basis using
paramagnetic/ferromagnetic iron albumin microspheres. The materials and
methods employed were as follows.
I. Preparation. A water-in-oil emulsion polymerization method was used to
prepare microspheres approximately 2-5 micron diameter consisting of
heat-denatured human serum albumin matrix in which Fe.sub.3 O.sub.4,
150-250 Angstrom in size is embedded. In the experiments described below,
the following preparation was used. An aqueous solution of 215 mg human
serum albumin, 72 mg magnetite in the form of an aqueous suspension
(Ferroflutics Corporation) was made in a volume of 1 ml distilled water. A
0.5 ml aliquot of this suspension was homogenized in 30 ml of cottonseed
oil by sonication for one minute. The homogenate was then added dropwise
to 100 ml of stirred (1600 RPM) cottonseed oil kept at a constant
temperature of 135.degree. C. At ten minutes the emulsion was removed from
the heat and stirred until cool. The microspheres were washed free of the
oil by centrifugation in anhydrous ether; they were washed free of ether
and resuspended in 0.1% Tween 80 and 0.15 normal saline. Microspheres were
suspended at a concentration of 10 mg/ml. Prior to use the microsphere
suspension was vigorously agitated without sonication. The uniformity of
size of the microspheres was checked under the microscope.
II. Rat Imaging Studies. Magnetic resonance imaging of rats and rabbits was
performed with a horizontal bore (8 cm) superconducting magnet system at a
magnetic field strength of 1.4 T, corresponding to a l.sub.H resonance
frequency of 61.4 MHz. Images were acquired using a two-dimensional
Fourier transform technique with slice selection determined by selective
irradiation. All images were obtained using 128 phase encoded gradient
steps. Reconstructed images (128.times.256 pixels) have a slice thickness
of 3 mm and submillimeter inplane resolution. To enhance T.sub.1 contrast,
an IR pulse sequence was employed with an echo time of 15 ms; inversion
time of 400 ms; and repetition time of 1,460 ms (IR 1,460/400/15).
Fasted (approximately 18 h) male Sprague-Dawley rates (approximately 400 g)
were anesthetized with intraperitoneal penobarbital (35 mg/kd) and
securely placed on a calibrated carrier and inserted into the magnet.
Tubes containing paramagnetically doped water or agar gels of known
T.sub.1 and T.sub.2 were placed alongside the animal. Baseline images were
obtained to optimize liver position within the imaging plane.
After baseline images, animals were removed from the magnet and injected
with Fe.sub.3 O.sub.4 albumin microspheres in aqueous suspension at doses
of 5-50 mg/microspheres 1,000 g animal weight, into the tail vein. Care
was taken not to alter the positioning of the animal during injection and
reinsertion into the magnet. Various T.sub.1 and T.sub.2 weighted pulse
sequences were utilized. Imaging was being immediately and continued for
1.5 to 3 h initially. Rats were subsequently imaged at 18 hrs, 1 month and
3 months.
III. Rat Biodistribution Studies. A group of 100-200 g male Sprague-Dawley
rats, fasted for approximately 18 h, were anesthetized with
intraperitoneal pentobarbital (35 mg/kg) and injected with serial
concentrations of magnetite-albumin microspheres through a tail vein. At
30 min, the animals were killed by cervical dislocation and tissues
obtained for T.sub.l and T.sub.2 analyses Samples included blood (obtained
by cardiac puncture), liver, spleen, kidney and thigh muscle (obtained by
excision). In all samples T.sub.1 and T.sub.2 were obtained within 45 min.
of death.
All T.sub.1 and T.sub.2 relaxation measurements were performed with an IBM
PC-20 Minispec pulse NMR spectrometer (IBM, Danbury, CT, U.S.A.). This
permanent magnet has a field strength of 0.47 T, corresponding to a .sup.1
H resonance frequence of 20 MHz and operating at 38.degree. C. A
microprocessor provided automatic calculations of T.sub.1 and T.sub.2.
IV. Rabbit VX2 Model. VX2 carcinoma was implanted in livers of New Zealand
white rabbits weighing 800 to 1500 g. by direct laparoscopic intrahepatic
implantation. This tumer reaches approximately 1 cm in size and creates
nodular metastases within the liver during the first three weeks after
implantation.
Tumor bearing animals and controls were studied to determine the
biodistribution of the magnetite albumin microspheres, 30 min. post
infusion. Animals were sacrified and liver sections obtained to
demonstrate histologic correlation with imaging findings, measure the
water content of the VX2 metastases and surrounding normal liver and to
determine relaxation times T.sub.1 and T.sub.2 in vitro. Water content was
measured by weighing specimens to a constant weight at 60.degree.. In
vitro spectroscopy was performed in the IBM PC-20 spectrometer. Liver,
tumor, spleen, muscle and blood were studied.
RESULTS
1. Biodistribution of Fe.sub.3 O.sub.4 Albumin Microspheres There is a near
linear increase in splenic T.sub.2 relaxivity (R.sub.2) with dose, peaking
at approximately 50 mg microspheres/Kg body weight with a 79% increase in
T.sub.2 relaxivity (R.sub.2) and no significant change in T.sub.l. At a
dose of 10 mg/Kg, there is a 41% increase in splenic T.sub.2 relaxivity
(R.sub.2) with no change in T.sub.1. There is a 60% increase in T.sub.2
relaxivity (R.sub.2) of normal liver at doses of 10 -20 mg microspheres
and a 16% increase in T.sub.1 relaxivity (R.sub.2). At 30 minutes post
injection of microspheres there is no evidence of microspheres in the
intravascular space with no change in blood and renal T.sub.1 and T.sub.2
parameters.
Peak hepatic (and splenic) superparamagnetic effect are seen within five
minutes post infusion based on subjective imaging criteria. There is
predicted 50-80% first pass clearance of particles this size with 40-70%
hepatic and 25-55% splenic uptake. A small percentage is taken up by bone
marrow and pulmonary macrophages. The ultimate fate of the
superparamagnetic albumin microspheres is not known. Several weeks post
infusion of a dose of 10 mg/microspheres/Kg body weight there is
persistent diminution of hepatic T.sub.2 but there is only a mild to
moderate decrease in T.sub.2 at 3 months. This probably reflects the
normal splenic and hepatic RES turnover which is estimated as 2-3 months
in the rat, and is unknown in humans.
2. Rabbit Tumor Model. Using a VX2 tumor model, a 65% reduction in T.sub. 2
and a 42% reduction in T.sub.l of normal liver is seen on spectroscopic
analysis post infusion of 10 mg of Fe.sub. 3O.sub.4 albumin
microspheres/Kg. There is a 13% reduction of T.sub. 2 of VX2 tumor at its
margin and a 3% reduction of T.sub.l, presumably representing small
patches of unreplaced normal parenchyma insinuated in tumor. At the tumor
margin there is a 53% increase in the difference between normal and
pathologic tissue T.sub.2 values at the tumor margin and a 39% increase in
the difference of T.sub.1 values at the tumor margin measured c
spectroscopy. Subjectively, the anatomic margins of normal and pathologic
tissues became more distinct, as did the true margin of liver, abdominal
wall and adjacent viscera, and small areas of tumor involvement became
more apparent. This increase in contrast was evident on shorter TR, TE and
mixed T.sub.1 and T.sub.2 pulse sequences with a single average. There was
no apparent loss of normal anatomic detail post infusion.
DISCUSSION
A. Toxicity/Fe Overload. There is more than a 20 fold margin of safety or
dosage "window" with a dose of 1400 mg of microspheres required to achieve
an Fe load with the low range of hepatic Fe toxicity of 1 mg Fe/g liver
wet weight. A 250 fold overdosage is necessary to load the liver with 10
mg Fe/g wet weight which is often seen in symptomatic transfusional
hemosiderosis (range 1-10 mg/g liver wet weight). The Fe content of normal
human liver is 0.15+0.02 mg Fe/g liver wet weight. The extrapolated adult
dosage of microspheres, based on 10 mg/Kg microsphere body weight is 700
mg of microspheres for an average 70 Kg adult, or 140 mg of magnetite (20%
of microsphere weight). With an average liver weight of 2200 g (range of
approximately 1700-2800 g) and 70% deposition in liver RES (98 mg,
Fe.sub.3 O.sub.4), an average single dose would transiently increase total
hepatic Fe by 0.044 mg/g liver.
B. Magnetic Properties. Fe+3 is a potent paramagnetic with 5 unpaired
electrons and a magnetic moment of 5.9 (Bohr magnetons). An increase in
T.sub.2 relaxivity (R.sub.2) in particulate bound form of paramagnetic
material such as iron (i.e., methemoglobin, hemosiderin, etc.) has been
observed. A pathophysiologic model of this phenomena is seen in
transfusional hemosiderosis and chronic parenchymal hemorrhage. However,
the T.sub.2 relaxivity seen with magnetite-albumin microspheres far
exceeds the apparent paramagnetic/ferromagnetic properties of hemosiderin
based on total Fe load necessary to achieve calculated T.sub.1 and T.sub.2
values. The reason for this is not known with certainty. Possibly the
maximum peripheral and central dispersion of magnetite in the albumin
matrix results in maximum generation of microfield inhomogeneity. The
diminution in T.sub.2 probably represents largely a T.sub.2.sup.* effect.
In addition, the enhanced T.sub.2 effect of iron (Fe) in the form of
particulate magnetite below 300 Angstroms size is probably due to its
superparamagnetic properties. The effect of the magnetic particles is that
abnormal tissues become intense (relative increase in signal intensity)
relative to normal tissue because of the action of the iron compound on
normal tissue. This is advantageous since hot spot imaging of
abnormalities is usually preferable to cold spot imaging.
Particulate iron less than 300 A in size such as magnetite is
superparamagnetic having a much larger magnetic susceptibility than
paramagnetic material. Magnetization increases with increased applied
external field from 0.3 to 0.9 Tesla and is rapidly lost when the
superparamagnetic species is removed from the external field. This low
remnance of residual magnetization prevents clumping or aggregation of
microspheres due to attraction between magnetized particles which would
adversely affect its biodistribution. The large magnetic moment of
superparamagnetic material generates local field inhomogeneities and
presumably promotes dephasing of proton spins and acceleration of
transverse relaxation. Magnetite therefore exhibits a different relaxation
mechanism than soluble paramagnetic contrast agents such as gadolinium
DTPA which show equal enhancement of T1 and T2 relaxation, obeying the
Bloemberger-Solomon equation.
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