SPECIFIC CONTRAST AGENTS FOR MAGNETIC RESONANCE IMAGING OF THE SMALL INTESTINE AND HEPATOBILIARY SYSTEM
This is a continuation-in-part of copending Serial No. 07/205,201, filed June 10, 1988.
The present invention is directed to a novel class of carboxylic acid derivatives of the iron (III) chelate of desferrioxa ine B which are useful as magnetic resonance imaging contrast agents for renal, hepatobiliary and, in particular, small intestine image enhancement.
This invention was made with Government support under Grant No. CAa8918 awarded by the Department of Health and Human Services. The Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
The iron (III) chelate of desferrioxamine B is a known magnetic resonance imaging contrast agent that gives both renal and hepatobiliary enhancement. It is one of the soluble paramagnetic compounds used for this purpose. Other soluble paramagnetic compounds, such as Gd-DTPA, suffer from various disadvantages, such as, rapid diffusion from the vascular system into the extracellular space, lack of specificity for liver and essentially no concentration in the bile. Manganese dichloride concentrates in the liver after IV injection.
is transported to the bile and excreted in the feces. However, it is toxic and is not practically useful in a clinical situation. Iron (III) complexes Fe-EHPG and Fe-HBED are useful contrast agents in the liver and are secreted into the bile. However, their appearance in the bile does not take place until approximately 20 minutes post injection and furthermore, enhancement of the lumen of the small intestine is seldom observed using these agents.
The magnetic resonance imaging of the gastrointestinal tract is limited by inherently low image contrast, motion and variable anatomic appearance. Agents for this purpose are normally administered orally and reside within lumen of the GI tract to provide either positive (increased signal strength) or negative (decreased signal strength) contrast. In the usual situation, positive contrast is provided by ingestion of paramagnetic agents .such as Gd-DTPA or iron citrate, while negative contrast is provided by displacement of the luminal proton-containing contents by gas (such as carbon dioxide from gas tablets) or perfluorocarbons. Negative contrast can also be obtained by ingestion of ferromagnetic or superpara agnetic particles, such as ferrite.
There is thus a need in the art for MRI contrast agents which are useful both for hepatobiliary studies and for gastrointestinal studies, particularly when introduced by intravenous administration, rather than by oral administration.
It is thus an object of the present invention to provide improved desferrioxamine B contrast agents for magnetic resonance imaging.
It is yet another object of the present invention to provide magnetic resonance imaging contrast agents having high stability, low toxicity, physiological tolerability and which provide for positive imaging of the small intestine rapidly after intravenous administration.
It is a further object of the present invention to provide desferrioxamine B contrast agents which are organ selective.
SUMMARY OF THE INVENTION
Briefly, these and other objects of the present invention are accomplished by providing chemically stable, physiologically tolerable desferrioxamine B derivatives for n vivo use for MRI imaging. The present invention provides a method for performing magnetic resonance imaging diagnostic procedures in a subject comprising the step of administering intravenously to the subject a magnetic resonance imaging enhancement effective amount of a diagnostic medium comprising the compound of the following formula in a physiologically acceptable carrier:
wherein X is carbon or nitrogen; R is -desferrioxamine B; j is an integer from 1 to n; k is an integer from 0 to m; s is an integer from 0 to p; p+n+m- is less than
or equal to 18; each Rι j is independently aryl of 6 to 14 carbon atoms, or substituted aryl having at least one substituent selected from the group consisting of halo, haloalkyl of 1 to 6 carbon atoms and 1 to 13 halo atoms, alkyl of 1 to 6 carbon atoms, nitro, amino, sulfonyl, hydroxyl, alkoxy of 1 to 6 carbon atoms, carboalkoxy of 2 to 6 carbon atoms, alkoxycarbonyl of 2 to 6 carbon atoms and sulfhydryl; and each R 2 J , R 3 k , R**, R 7 ' and R g * is independently hydrogen, aryl or substituted aryl as defined above; and R, is hydroxy, linear or branched alkoxy of 1 to 18 carbon atoms, amino, mono or di- substituted amino, aryloxy or substituted aryloxy as defined above.
BRIEF DESCRIPTION OF THE DRAWING FIGS. 1A through IE are graphs showing the distribution of 59 Fe in labelled contrast agents, DF, GDF, PSDF, SDF and PGDF, respectively.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Media according to the present invention are useful for magnetic resonance imaging diagnosis. Production of the diagnostic media according to the present invention may be performed according to ways known in the art, i.e., by forming a complex salt as described hereinbelow, optionally with the addition of customary additives such as galenicals, which may be suspended or dissolved in an aqueous medium followed by sterilization. Suitable additives include, for example, physiologically biocompatible buffers such as tromethamine hydrochloride, complexing agents such as diethylenediiaminepentaacetic acid, or if necessary electrolytes such as sodium chloride.
The compounds according to the present invention which are utilized in the present method are those of the following formula I:
wherein X is carbon or nitrogen; R is -desferrioxamine B; j is an integer from 1 to n; k is an integer from 0 to m; s is an integer from 0 to p; p+n+m is less than or equal to 18; each Rj j is independently aryl of 6 to 14 carbon atoms, or substituted aryl having at least one substituent selected from the group consisting of halo, haloalkyl of 1 to 6 carbon atoms and 1 to 13 halo atoms, alkyl of 1 to 6 carbon atoms, nitro, amino, sulfonyl, hydroxyl, alkoxy of 1 to 6 carbon atoms, carboalkoxy of 2 to 6 carbon atoms, alkoxycarbonyl of 2 to 6 carbon atoms and sulfhydryl; and each R 2 j , R 3 k , R**, R 7 * and R 8 * is independently hydrogen, aryl or substituted aryl as defined above; and R, is hydroxy, linear or branched alkoxy of 1 to 18 carbon atoms, amino, mono or di- substituted amino, aryloxy or substituted aryloxy as defined above.
Exemplary aryl groups are phenyl, substituted phenyl, biphenyl, substitutedbiphenyl, phenanthryl, anthracyl, substituted phenanthryl. A more preferred aryl group or aryloxy group are phenyl and phenoxy and preferably para substituted phenyl or phenoxy.
A second class of complexes are those comprising a metal ion selected from the group consisting of Fe (III) ,
Dy (III) , Gd (III) , Cr (III) and Mn (II) and a ligand of the formula (III)
wherein R is -desferrioxamine-B; t and u are integers from 0 to 3; R§ is hydroxy or an alkoxy of 1 to 18 carbon atoms or an aryloxy of 6 to 14 carbon atoms, or substituted aryloxy having at least one substituent selected from the group consisting of aryl, nitro, sulfonyl, hydroxl, alkoxy of 1 to 6 carbon atoms, carboalkoxy of 2 to 6 carbon atoms, alkoxy carbonyl of 2 to 6 carbon atoms and sulfhydryl.
Components of the above formula may be made by treating a salt of desferrioxamine B, such as the mesylate, with an appropriate anhydride of an aromatic dicarboxylic acid, such as phthalic anhydride. By using two or more equivalents of the anhydride to one equivalent of the protected desferrioxamine B, in a basic medium, the primary amino group of the desferrioxamine B will react with the anhydride and form an N-carboxylate desferrioxamine B derivative. Any -O- esters that may have formed during the amidation reaction may. then be removed under conventional conditions to result in the N-carboxylated desferrioxamine B contrast agents according to the present invention. Treatment of the anhydride with desferrioxamine B will normally be conducted at around 100 to 110°C in a suitable solvent,
such as pyridine. Treatment of the ligand with an appropriate metal salt of the desired metal counterion will form the metal complex. Alternatively, the NOH groups may be protected by esterification or by complexation with a metal ion.
The preferred iron (III) complex of the N-carboxylated desferrioxamine B compound may be prepared by treatment thereof with ferric chloride in an acidic aqueous medium, followed by basification to a pH of about 7.5 to 8.
In the compounds according to the present invention the radical desferrioxamine B is of the following formula II
H
I
—N 00-NH • CO-KH /
<£ H 2>5 (CH 2 ) 2 (CH 2 ) 5
\ / \
N C N—C l II I II
HO O HO O
and is complexed with a paramagnetic ion.
The active ingredient of the contrast agents according to the present invention are generally prepared by treating a salt of desferrioxamine B, such as the mesylate, with an appropriate anhydride of a dicarboxylic acid. By using two or more equivalents of the anhydride to one equivalent of the protected desferrioxamine B, in a basic medium, the primary amino group of the desferrioxamine B will react with the anhydride and form an N-carboxylate desferrioxamine B derivative. Any -0- esters that may have formed during the amidation reaction may then be removed under conventional conditions to result in the N-carboxylated; desferrioxamine B contrast agents according to the
present invention. Treatment of the anhydride with desferrioxamine B will normally be conducted at around 100 to 110°C, in a suitable solvent, such as pyridine. Treatment of the ligand with an appropriate metal salt of the desired metal counterion will form the metal complex. Alternatively, the N-OH groups may be protected by esterification or by complexation with a metal ion.
The preferred iron (III) complex of the N-carboxylated desferrioxamine B compound may be prepared by treatment thereof with ferric chloride in an acidic aqueous medium, followed by basification to a pH of about 7.5 to 8.
Particularly preferred contrast agents according to the present invention are the complexes with iron (III) wherein n=l, m=l, p=l, R g , R 7/ R 2 , R 3 and R 3 are hydrogen, X is carbon, ! is phenyl or substituted phenyl and Rø is hydroxy or methoxy. Preferred ligands are shown in Table 1. In particular, the N-(3-phenylglutaramido) desferrioxamine B iron (III) complex is advantageously used for MRI imaging of the small intestine.
TABLE 1
PREFERRED LIGANDS
FORMULA I
TABLE 1 (Continued)
Other preferred contrast agents are the Iron (III) complexes with the ligands shown in Table 2.
TABLE 2
FORMULA III
R=desferrioxamine B
In addition, certain of the N-carboxylated desferrioxamine B ligands and complexes thereof with metals according to the present invention having the following formula are novel.
wherein X is carbon or nitrogen; R is -desferrioxamine B; j is an integer from 1 to n; k is an integer from 0 to m; s is an integer from 0 to p; p+n+m is less than or equal to 18; each Rj j is independently aryl of 6 to 14 carbon atoms, or substituted aryl having at least one substituent selected from the group consisting of halo, haloalkyl of 1 to 6 carbon atoms and 1 to 13 halo atoms, alkyl of 1 to 6 carbon atoms, nitro, amino, sulfonyl, hydroxyl, alkoxy of 1 to 6 carbon atoms, carboalkoxy of 2 to 6 carbon atoms, alkoxycarbonyl of 2 to 6 carbon atoms and sulfhydryl; and each R 2 j , R 3 k , R^, R 7 * and R 8 ' is independently hydrogen, aryl or substituted aryl as defined above; and Rj is hydroxy, linear or branched alkoxy of 1 to 18 carbon atoms, amino, mono or di- substituted amino, aryloxy or substituted aryloxy as defined above.
The contrast agents according to the present invention are particularly advantageous in that upon intravenous injection, they concentrate in bile, and in some cases, appear in the small intestine very rapidly, usually within about two minutes post injection. They are
useful in that they may be administered by the intravenous route, thus the entry into the small intestine is by the hepatobiliary system. Therefore, in addition to contrast enhancement of alimentary tract lumen, with its resulting increased anatomical information, the contrast agents also provide functional information. For example, hepatobiliary dysfunctions, such as bile duct occlusion will retard, lessen or prevent the transport of the contrast agent and thus be revealed by MRI. Appearance in the intestinal lumen, usually within two minutes post injection, is particularly advantageous for imaging since the optimum imaging time for the small intestine can thereby be readily predicted. Such timing is extremely difficult with use of orally administered agents for MRI of the small intestine. The contrast agents according to the present invention not only show specificity for the biliary system and small intestines, but they also are relatively non-toxic and substantially completely excreted. Conventional hepatobiliary contrast agents, such as Gd-DTPA, distribute in the extracellular volume and are not useful therefore for imaging the small intestine.
The reagents according to the present invention therefore can reveal additional details regarding the anatomy of the small intestines, and because of their very rapid localization in the bile and gut lumen, the MRI of these organ systems are simplified and thereby expedited.
It will be appreciated that other metal ions besides iron (III) may be utilized to complex with the ligands according to the present invention for use in magnetic resonance imaging. Useful metal ions include Dy (III), Gd (III) , Cr (III) , and Mn (II) .
The agents according to the present invention are utilized preferably in solutions made by addition of a solvent such as water, serum, albumin solutions, or saline to the complex. A typical injectable composition will contain about 10 mg/ml human serum albumin with a metal complex concentration of about 20 to 200 mg/ml. A 0.1 m phosphate buffer (7.5 pH) may be employed containing, for example, about 0.9% sodium chloride. The pH of the aqueous solutions may range between about 5 to 9 over a volume range of about 5 to 150 ml. This viscous diagnostic reagent may then be injected into a subject usually in an amount of .001 to 5 mmole of contrast agent, and the subject may then be imaged by a conventional magnetic resonance imaging system. Typical imaging systems are disclosed, for example in Scientific American. May 1982, pp. 78-88, and "NMR: A Primer For Medical Imaging" by Wolf and Popp, Slack Book Division (ISBN 0-943432-19-7) , which are incorporated herein by reference.
The following examples are provided by way of illustration of the invention but are not in any way intended to limit the invention.
EXAMPLE 1 Preparation of 3-Phenylσlutaric Anhydride 3 Phenylglutaric anhydride, 5.0 g (24 mmoles), was dissolved in 25 ml of dry acetic anhydride, and the resulting solution was heated to 100°C and stirred for 3 hours. The reaction mixture then was allowed to cool to 35°C and held at that temperature for i.5 hours. Removal of the acetic anhydride using a rotary evaporator, andrecrystallization of theresulting crude solid from chloroform gave 2.25 g (11.8 mmoles; 50% of theory) of the title compound, mp. 102-103 β C.
EXAMPLE 2 Preparation of N-f-phenylσlutaramidoldesferrioxamine To 1.5 g (2.28 mmoles) of desferrioxamine B esylate in 30 mL of pyridine was added 0.85 g (4.47 mmoles) of 3- phenylglutaric anhydride. The resulting solution was stirred for 2 hours at 110 β C, cooled to 55 β C, and then held at that temperature for 18 hours. Then 30 ml of 2 N aqueous ammonia was added to the reaction mixture, and it was stirred for 15 minutes at ambient temperature. Pyridine was removed from the reaction mixture by extraction with dichloromethane. The aqueous fraction then was striped to dryness using a rotary evaporator. The resulting solid residue was redissolved in 40 ml of water, and this solution was acidified to pH 2-2.5 by the addition of 1 N HCl. After acidification, the solution was filtered, and the filtrate was treated with an equivalent volume of acetone. Cooling of this solution afforded 1.29 g (1.72 mmoles; 75% of theory) of white solid, mp. 123.5-126°C. Anal. Calc'd for C 36 H 58 N 6 O n :C, 57.60; H, 7.73; N, 11.20. Found: C, 57.39; H, 7.59; N, 11.2. Mass spectrum (LSIMS) : (M+H) + =751; (M+Na) + =773.
EXAMPLE 3 Preparation of N-fGlutaramido)desferrioxamine B N-(glutaramido)desferrioxamine B was prepared similarly using 1.41 g (12.37 mmoles) of glutaric anhydride and 4.91 g (7.47 mmoles) of desferrioxamine B mesylate. Recrystallization from dilute HCl (pH 2.5) gave 3.08 g (4.6 mmoles, 62% of theory) of white solid, mp. 154- 158 β C. Anal. Calc'd for C^H^N β O,-: C, 53.41; H, 8.01; N, 12.46. Found: C, 53.29; H, 7.88; N, 12.37. Mass spectrum (LSIMS): (M+H) + «675; (M+Na) + »697.
EXAMPLE 4 Synthesis of N-(succinaτnido)desferrioxamine B N-(succinamido)desferrioxamine B was prepared similarly to the glutaric derivative using 0.68 g (6.8 mmoles) of succinic anhydride and 2.0 g (3.0 mmoles) of desferrioxamine B mesylate. Recrystallization from dilute HCl (pH 2.5) gave 0.72 g (1.1 mmoles, 37% of theory) of white solid, mp. 152-154°C. Anal. Calc'd for C 2 H 32 N 6 °n : C, 52.71; H, 7.93; N, 12.72. Found: C, 52.5; H, 7.9; N, 12.4. Mass spectrum (LSIMS): (M+H) + =661; (M+Na) + =683.
EXAMPLE 5
Preparation of the Feflll) Complex of N-(3- phenylσlutaramido)desferrioxamine fFe-PGDF) To 160 mg (12 mmoles) of imidoacetic acid dissolved in 1 ml of aqueous NaOH (pH=8) , was added 134 mg (5 mmoles) of FeCl 3 6 H 2 0. This solution was brought to pH=8 with 2N NaOH, and to it was added 375 mg (5 mmoles) of N-(3- phenylglutaramido)desferrioxamine (PGDF) dissolved in 1 mL of water. The resulting mixture was adjusted to pH-7.5 with 2N NaOH, and filtered through a 0.2 um filter to yield a red solution of the Fe(III) complex. After appropriate dilution with water, this material was used for in vivo imaging experiments. Similar Fe(III) complexes were prepared using the ligands made in Examples 3 and 4.
EXAMPLE 6 A normal, male Sprague-Dawley rat was anesthetized with 50 mg/kg i.p. sodium pentobarbital and fitted with a tail-vein catheter. The animal was then placed in an imaging coil, and the coil plus animal was inserted into the magnet bore of a General Electric Model CSI-II Magnetic Resonance Imager/Spectrometer. Non-gated spin- echo images were acquired using TR and TE equal to 320 and 15 msec, respectively. Following these pre-contrast
images, 0.52 mL of a 0.10 M Fe-PGDF solution was injected, and images were acquired at various time intervals post injection. Within one minute of injection the liver, stomach and small intestine showed enhanced contrast, particularly the small intestine. One hour post injection contrast enhancement of the urinary bladder began with the contrast enhancement of the small intestine being diminished. Several hours post injection showed only enhancement of contrast in the urinary bladder.
EXAMPLE 7 Using the basic procedure of Examples 1, 2 and 5, Fe (III) complexes were prepared except that 3-phenylglutaric anhydride was replaced . by, respectively, p-chlorophenylglutaric anhydride, p-isopropylphenylglutaric anhydride, p-t-butylphenyl glutaric anhydride, p-nitrophenylglutaric anhydride, p-trichloromethylphenylglutaricanhydride, andphthalic anhydride.
EXAMPLE 8 "
Fe-PGDF, 0.25 g (0.31 mmol) was dissolved in 10 mL of anhydrous methanol, and the resulting red solution was treated with 5 mL of a 40% w/w solution of BF 3 in methanol (24 mmol) . The solution became purple in color as the BF 3 was added. The reaction mixture was warmed to 60°C for 15 minutes. The methanol then was removed using a rotary evaporator. The residual purple liquid then was treated with aqueous pH 7 buffer, and polystyrene-divinyl benzene beads (e.g., "BioBeads," BioRad, Inc., Hercules, CA) . The Fe-PGDF methyl ester was adsorbed onto the beads. Salts and by-products were removed by washing the beads with water. The Fe-PGDF methyl ester was recovered by extraction with methanol.
EXAMPLE 9
A solution of p-N0 2 -PGDF (1.5 g 0.188 mmol) in methanol
(25 mL) along with 10% Palladium-on-carbon catalyst
(0.10 g) was placed under a hydrogen atmosphere (1 atmosphere pressure) . After being stirred for five hours at 25 β C, the reaction mixture was filtered to remove the catalyst. The resulting filtrate was concentrated under reduced pressure to a volume of 1-2 L. Upon cooling to -20"C, p-NH 2 -PGDF was isolated as a white solid, 0.117 g (0.155 mmol).
EXAMPLE 10 The distribution of 5 Fe in the whole body, urine and feces of rats was followed for seven days after i.v. bolus doses of 59 Fe-labelled substituted ferriόxamine contrast agents. The results are shown in FIGS. 1A-1E. DF=desferrioxamine; GDF=N-(glutaramido) desferrioxamine; PSDF=N- (phenylsuccinamido) -desferrioxamine; SDF=N-(succinamido)desferrioxamine. Each animal and its excreta were counted at selected time points for seven days following injection. For 5 Fe-DF, 59 Fe-SDF, and 59 -Fe-GDF, nearly all of the dose was excreted in the urine within the first day. With the addition of a phenyl group (i.e. , 5 Fe-PSDF and 59 Fe-PGDF) , nearly half of the total radiolabel appeared in the feces, demonstrating hepatobiliary elimination. In all cases, little of the injected 59 Fe remained in the body at 7 days. Data are means plus standard deviations (n=3,4) .
