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Description  |
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BACKGROUND OF THE INVENTION
The present invention relates to NMR imaging of living subjects. More
specifically, it relates to agents which can be used to enhance NMR
contrast in such subjects.
Nuclear magnetic resonance (NMR) has been used for many years as a means of
chemical analysis. NMR is a type of radio frequency spectroscopy which is
based upon small energy differences between electrically charged atomic
nuclei which are spinning parallel or antiparallel to an applied magnetic
field. When radio frequency energy is applied to the sample, these
spinning atomic nuclei change spin states and in doing so, absorb some of
the radio frequency energy. Nuclei in slightly different chemical
environments within the same molecule change spin state at slightly
different energies and this produces characteristic absorptions or
resonances which help identify the molecular structure.
NMR has more recently been used in examinations of the human body. Other
methods such as computerized axial tomography (CAT scanning) have been
used in the past for this purpose, and still are. However, because NMR
does not use ionizing radiation, it is believed to have some safety
advantages over CAT. Thus, NMR is an advantageous method of producing
cross-sectional images of the human body.
The quality of the images obtained from an NMR scan are based on two
properties: the proton densities of the various tissues and differences in
proton relaxation rates. The proton density of tissues cannot be readily
altered. Proton relaxation rates can be adjusted by adding a paramagnetic
relaxation agent, more commonly known as a "contrast agent." Contrast
agents enhance the contrast in NMR images between magnetically similar but
histologically dissimilar tissues.
Gadolinium has been tested as a contrast agent in the past because it has a
large magnetic moment, which efficiently relaxes magnetic nuclei.
Gadolinium's strong paramagnetic properties are the result of its seven
unpaired electrons.
One drawback of gadolinium as a contrast agent is its toxicity to animals.
One possible remedy for this problem is to incorporate gadolinium in a
compound that would pass through the body and be excreted without
releasing toxic gadolinium ions. Unfortunately, the rare earth elements,
such as gadolinium, do not form stable covalent bonds with organic
molecules, so such molecules can decompose in vivo and release the toxic
ions. Complexes of gadolinium might overcome this problem.
There is a need for effective contrast agents which avoid the toxicity
problems inherent in using gadolinium.
SUMMARY OF THE INVENTION
The present invention concerns NMR contrast agents which include a chelate
of gadolinium with either 1,4,7-triazacyclononane-N,N',N"-triacetate
(NOTA), 1,4,7,10-tetrazacyclododecane-N,N',N",N'" tetracetate (DOTA), or
1,5,9-triazacyclododecane-N,N',N"-triacetate (DOTRA), or salts thereof.
When the phrase "salts thereof" is used in this patent, it means that one
of the acidic hydrogen ions on an acetate group has been replaced by
another cation, not that an entire acetate group has been replaced. The
particular juxtaposition of the nitrogen and oxygen atoms has an important
effect on the chelating properties of NOTA, DOTA, and DOTRA, so removal of
an entire acetate group would harm that property. Of course, upon
dissolving the chelate in solution, the cation that has replaced a
hydrogen ion would dissociate leaving the same central ionic species.
These contrast agents can be used to enhance NMR contrast in a living
subject by administering internally to the subject an effective amount of
the agent. "Administering internally" is intended to include methods such
as injection, ingestion, or the like which would be known to one skilled
in this field.
DOTRA, DOTA, and NOTA reduce or prevent the toxic effects of the Gd.sup.3+
cation to in vivo processes by firmly complexing with it. DOTRA and DOTA
form gadolinium chelates that are especially stable, with NOTA binding
somewhat less firmly, possibly due to the small size of the "hole" in
NOTA's molecule.
This binding strength should result in very low biological toxicity for
contrast agents in accordance with the present invention. In addition, the
agents appear to have substantially better relaxation properties than some
prior art agents, which will permit the use of a smaller amount of the
agents to achieve the same effect.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The following is a procedure which can be used to synthesize NOTA:
Step 1: Synthesis of N,N',N"-tri(p-toluenesulfonyl)diethylene-triamine
A solution of p-toluenesulfonyl chloride (191 g) in ether (500 ml) was
added drop by drop to a solution of diethylene triamine (38 ml) and sodium
hydroxide (40 g) in water (250 ml). The mixture was stirred for one hour
at room temperature. A white precipitate was formed and was collected by
filtration, washed with water, and then recrystalized using methanol.
(Acetonitrile could also be used). The melting point of the recrystalized
precipitate was 174.degree. to 175.degree. C. The yield was 90%, and
should always be above 70%.
Step 2: Synthesis of di(p-toluenesulfonyl)ethylene glycol
Twenty-eight ml of ethylene glycol and 100 ml of pyridine were added over a
2.5 hour period to a stirred mixture of tosyl chloride (210 g) in pyridine
(225 ml), with the mixture being cooled by a water bath. After stirring
for several hours, the mixture was shaken with one liter of ice water for
about ten minutes and then filtered. The residue was washed with ether,
dilute sulfuric acid, water, and finally ether. (Each of the washed
liquids was ice cold.) The residue was then dried by vacuum pumping and
recrystallized from boiling acetonitrile. The recrystallized residue,
yield 75%, had a melting point of 123.degree. to 125.degree. C.
Step 3: Preparation of the disodiom salt of
N,N',N"-tri(p-toluenesulfonyl)diethylene triamine
Each part of this step was conducted under a nitrogen atmosphere. 2.65
grams of sodium metal was weighed in hexane and placed in about 75 ml of
pure ethanol. The sodium-ethanol reaction is highly exothermic, and the
heat helps dissolve the sodium to give sodium ethoxide. A hot slurry of
1,4,7-tritosyl-1,4,7-triazaheptane (28.3 g) from Step 1 and 150 ml of
ethanol was stirred in a reaction vessel with a reflux condenser. The
slurry was heated to reflux using an oil bath, and then the sodium
ethoxide was added as rapidly as possible. After continued stirring and
flushing with nitrogen, a white solid precipitated. Slight heating and
flushing continued until all the ethanol was removed and the dry disodium
salt of 1,4,7-tritosyl-1,4,7-triazaheptane was left.
Step 4: Synthesis of 1,4,7-triazacyclononane-N,N',N"-tritosylate
This step was conducted without removing the dry salt from the Step 3
reaction vessel. The dry disodium salt was dissolved in 225 ml of dry
dimethyl formamide (DMF), once again under a nitrogen atmosphere. The
mixture was stirred and heated to 95.degree. to 110.degree. C. Next, a
0.2M solution of ethylene glycolditosylate (18.5 g) in DMF was added over
a period of three hours. After one additional hour of stirring at
100.degree. C., the mixture was cooled overnight. It was then concentrated
by distillation under reduced pressure until precipitation began. The
concentrate was poured into 500 mls of vigorously stirred water and
filtered. The residue was washed with water, dried by vacuum pumping, and
recrystallized from boiling acetone. The product,
1,4,7-triazacyclononane-N,N',N"-tritosylate, had a melting point of
217.degree. to 220.degree. C. and was present in a yield of 70%.
Step 5: Synthesis of 1,4,7-triazacyclononane trihydrobromide
One hundred twenty ml of a mixture of 47% HBr, 67 ml of glacial acetic
acid, 13.99 g of the product of Step 4 were heated to 100.degree. C., and
the volume was then remeasured. The mixture was then refluxed for fifty
hours and concentrated by atmospheric distillation to about 20% of the
beginning volume. The concentrate was then filtered. The residue,
containing 1,4,7-triazacyclononane-N,N',N"-trihydrobromide, was extracted
into water and then recovered by evaporation in vacuo. The trihydrobromide
was recrystallized from boiling hydrobromic acid. Its melting point was
280.degree. C, and it was present in 70% yield. Tosylate groups were
completely absent in the NMR spectra of the trihydrobromide.
Step 6: Synthesis of 1,4,7-triazacyclononane-N,N',N"-triacetete (NOTA)
A solution of 4.72 g of bromoacetic acid and 1.2 g of sodium hydroxide in
10 ml of water was added with stirring to a solution of 3.72 g of the
product of Step 5 and 1.2 g of sodium hydroxide in 3.5 ml of water at
about 20.degree. C. The mixture was heated to 85.degree. C. with an oil
bath while being stirred, and then 1.2 g of sodium hydroxide, dissolved in
6.5 ml of H.sub.2 O, was added dropwise with stirring. The temperature was
maintained between 80.degree. to 90.degree. C. for one and one-half hours.
The contents of the flask were then cooled to room temperature and the pH
was adjusted to about 3.5 with concentrated hydrobromic acid. 25 ml of
ethyl alcohol was added, and the solution was stirred for an hour under
refrigeration. A white crystalline precipitate formed which was filtered
out, washed with pure ethanol, and dried in a vacuum oven at 70.degree. C.
overnight. This product was NOTA, and the 2.5 g of it represented an at
least 70% yield.
Elemental analysis showed close correspondence to what was expected for
C.sub.12 H.sub.21 O.sub.6 N.sub.3 (NaBr).sub.3.3H.sub.2 O. Calculated:
25.58% C, 4.79% H, 7.46% N, 28.42% Br, and 8.17% Na. Found: 25.39% C,
4.89% H, 7.43% N, 28.44% Br, and 8.00% Na.
This synthesis can be summarized as shown below.
##STR1##
DOTA and DOTRA can be synthesized using generally the same procedure, but
starting with triethylene tetraamine instead of diethylene triamine to
synthesize DOTA and dipropylene triamine and 1,3 propanediol instead of
diethylene triamine and ethylene glycol, respectively, for synthesizing
DOTRA. The remaining reagents would be identical with only the
stoichiometric quantities varying for DOTA.
Once DOTA, NOTA or DOTRA has been obtained in crystalline form, a measured
amount of it is dissolved in water and an equimolar amount of a gadolinium
salt, such as gadolinium chloride or gadolinium nitrate, is added to the
solution. The Gd-NOTA complex forms spontaneously above pH 5 while the
Gd-DOTA and Gd-DOTRA complexes are kinetically slower to form and may
require heating to 80.degree. C. for 30 minutes to increase the rate of
chelation.
Salts of DOTA, NOTA and DOTRA could, of course, also be used, since the
counter ions will dissociate in solution. What synthetic procedure is most
convenient may dictate which salt to use. The meglumine salt of Gd-DOTA,
Gd-NOTA or Gd-DOTRA is one which should be useful in contrast agent
formulations.
The contrast agents could be formulated as a saline solution and packaged
in bottles having a rubber septum across the opening to permit withdrawing
the solution with a syringe.
Contrast agents in accordance with the present invention can be used with
NMR apparatus which are well known to those skilled in this field.
Examples of U.S. patents which disclose NMR apparatus are U.S. Pat. Nos.
4,374,360; 4,398,148; 4,409,550; 4,425,547; 4,442,404; and 4,450,408, all
of which are incorporated herein by reference. NMR imaging should probably
be done within a few hours after administering the contrast agent to the
subject, since the agent should be excreted from the body fairly rapidly.
The preceding is intended to illustrate specific embodiments of the present
invention, and not to be an exhaustive description of all possible
embodiments. Those skilled in this field will recognize that certain
modifications could be made.
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