U.S. patent number 5,196,348 [Application Number 07/725,692] was granted by the patent office on 1993-03-23 for perfluoro-crown ethers in fluorine magnetic resonance spectroscopy of biopsied tissue.
This patent grant is currently assigned to Air Products and Chemicals, Inc.. Invention is credited to Joseph A. Rubertone, Frank K. Schweighardt.
United States Patent |
5,196,348 |
Schweighardt , et
al. |
* March 23, 1993 |
Perfluoro-crown ethers in fluorine magnetic resonance spectroscopy
of biopsied tissue
Abstract
A method is disclosed for nuclear magnetic resonance
spectroscopy wherein the improvement is the use of perfluoro
15-crown-5 ether which has 20 magnetically similar fluorine
providing a superior signal to noise ratio with resultant enhanced
diagnostic resolution.
Inventors: |
Schweighardt; Frank K.
(Allentown, PA), Rubertone; Joseph A. (Coatesville, PA) |
Assignee: |
Air Products and Chemicals,
Inc. (Allentown, PA)
|
[*] Notice: |
The portion of the term of this patent
subsequent to June 13, 2006 has been disclaimed. |
Family
ID: |
27064955 |
Appl.
No.: |
07/725,692 |
Filed: |
July 3, 1991 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
535818 |
Jun 11, 1990 |
5068098 |
|
|
|
Current U.S.
Class: |
436/173; 514/450;
514/832 |
Current CPC
Class: |
A61K
49/06 (20130101); G01R 33/281 (20130101); G01R
33/465 (20130101); G01R 33/5601 (20130101); Y10S
514/832 (20130101); Y10T 436/24 (20150115) |
Current International
Class: |
A61K
49/06 (20060101); G01R 33/44 (20060101); G01R
33/465 (20060101); G01R 33/28 (20060101); G01N
024/00 (); G01N 031/00 (); A61K 031/335 () |
Field of
Search: |
;424/9 ;436/173
;128/653.4 ;514/450,832 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Bottomley, Pa., Computerized Radiology 8(2):57-77 (1984). .
Nature, vol. 242, pp. 190-191, 1973 Lauterbur, PC. .
Damadian, Science, vol. 171, p. 1151, 1971 Tumor Detection by
Nuclear Magnetic Resonance. .
Mattrey, R. F.; "Perfluorocarbon Compounds:Applications in
Diagnostic Imaging"; SPIE; vol. 626, Medicine, XIV/Pacs IV (1986),
pp. 18-23. .
Longmaid, III et al.; "In vivo .sup.19 F NMR Imaging of Liver,
Tumor and Abscess in Rats"; Investigative Radiology; Mar./Apr.
1985; vol. 20; pp. 141-144. .
Patronas et al; "Brain-tumor Imaging Using Radiopaque
Perfluorocarbon"; Journal of Neurosurgery; May 1983; vol. 58; pp.
650-653. .
Mattrey, R. F. et al; "Perfluorochemicals as US Contrast Agents for
Tumor Imaging and Hepatosplenography:Preliminary Clinical Results";
Radiology; May 1987; vol. 163; No. 2. pp. 339-343. .
Parhami & Fung; "Fluorine-19 Relaxation Study of Perfluoro
Chemicals as Oxygen Carriers"; J. Physical Chemicals; 1983; pp.
1928-1931. .
Nunnally, et al; "Fluonine-19 (.sup.19 F) NMR in Vivo:Potential for
Flow and Perfusion Measurements"; Proceedings of the Society and
Magnetic Resonance in Medicine; 2nd Annual Meet.; Aug. 16-19; pp.
226. .
Reid, et al; "The Influence of Oxygenation on the .sup.19 F
Relaxation Rates in Flusol-da"; Phys. Med. Biol; vol. 30; No. 7;
pp. 677-686 (1985). .
Wyrwics et al; "Observations of Fluorinated Anesthetics in Rabbit
Brain by .sup.19 F NMR"; Proceedings of the Soc. of Magnetic
Resonance in Med; 2nd Annual Meet; Aug. 16-19; (1983); pp. 381-382.
.
Clark et al; "Perfluorinated Organic Liquids and Emulsions as
Biocompatible NMR Imaging Agents for .sup.19 F and Dissolved
Oxygen"; Adv. Exp. Med. Biol.; vol. 180(6); pp. 835-845;
1984..
|
Primary Examiner: Hollrah; Glennon H.
Assistant Examiner: Hollinden; Gary E.
Attorney, Agent or Firm: Chase; Geoffrey L. Simmons; James
C. Marsh; William F.
Parent Case Text
This is a division, of application Ser. No. 07/535,818 filed Jun.
11, 1990 now U.S. Pat. No. 5,068,098.
Claims
We claim:
1. In a method of obtaining a .sup.19 F-fluorine magnetic resonance
spectrum from body organs or tissue by administering to a mammal a
fluorine-containing agent in a diagnostically effective amount to
provide a fluorine magnetic resonance spectrum from such organs or
tissues, the improvement comprising using as said
fluorine-containing agent an aqueous isotonic emulsion of perfluoro
15-crown-5 ether and after the administration of the agent to the
mammal, the desired tissue is biopsied and the biopsied and
extracted tissue is analyzed to give an .sup.19 F-fluorine magnetic
resonance spectrum.
2. The method of claim 1 where in the concentration of the emulsion
is the range of 5 to 25 wt % of the perfluoro 15-crown-5 ether.
3. The method of claim 1 wherein said fluorine-containing agent is
administered within an organ, tissue, space, blood vessel or cavity
of a mammal.
4. The method of claim 1 wherein the fluorine-containing agent is
administered by a process selected from the group consisting of
direct injection into a body organ, direct injection into a body
cavity, direct injection into a tissue area.
Description
TECHNICAL FIELD
The present invention is directed to magnetic resonance
spectroscopy, also referred to as nuclear magnetic resonance
spectroscopy. More particularly, the present invention is directed
to methods and compositions for improving magnetic resonance
spectra of body organs and tissues using fluorochemicals having
unexpectedly enhanced signal to noise response ratios and having an
unexpectedly high and diagnostically useful NMR signal response to
the presence of oxygen.
BACKGROUND OF THE PRIOR ART
The recently developed techniques of MRI (magnetic resonance
imaging) or NMR (nuclear magnetic resonance) imaging encompasses
the detection of certain atomic nuclei utilizing magnetic fields
and radio-frequency radiation. It is similar in some respects to
X-ray computed tomography (CT) in providing a cross-sectional
display of the body organ anatomy with excellent resolution of soft
tissue detail. In current use, the images produced constitute a map
of the distribution density of protons and/or their relaxation
times in organs and tissues. The MRI technique is advantageously
non-invasive as it avoids the use of ionizing radiation.
While the phenomenon of NMR was discovered in 1945, it is only
respectively recently that it has found application as a means of
mapping the internal structure of the body as a result of the
original suggestion of Lauterbur (Nature, 242, 190-191, 1973). The
lack of any known hazard associated with the level of the magnetic
and radio-frequency fields that are employed renders it possible to
make repeated scans on vulnerable individuals. Additionally, any
scan plane can readily be selected including transverse, coronal,
and sagittal sections.
In an NMR experiment, the nuclei under study in a sample (e.g.
protons) are irradiated with the appropriate radio-frequency (RF)
energy in a highly uniform magnetic field. These nuclei, as they
relax, subsequently emit RF radiation at a sharp resonant
frequency. The emitted frequency (RF) of the nuclei depends on the
applied magnetic field.
According to known principles, nuclei with appropriate spin when
placed in an applied magnetic field [B, expressed generally in
units of gauss or tesla (10<4>gauss)] align in the direction
of the field. In the case of fluorine, these nuclei precess at a
frequency f=94.08 MHz at a field strength of 2.35 Tesla. At this
frequency, an RF pulse of radiation will excite the nuclei and can
be considered to tip the nuclei out of the field direction, the
extent of this rotation being determined by the pulse duration and
energy. After the RF pulse, the nuclei "relax" or return to
equilibrium with the magnetic field, emitting radiation at the
resonant frequency. The decay of the signal is characterized by two
relaxation times, i.e., T1, the spin-lattice relaxation time or
longitudinal relaxation time, that is, time taken by the nuclei to
return to equilibrium along the direction of the externally applied
magnetic field, and T2, the spin-spin relaxation time associated
with the dephasing of the initially coherent precession of
individual proton spins. These relaxation times have been
established for various fluids, organs and tissues in different
species of mammals.
In MRI, scanning planes and slice thickness can be selected without
loss of resolution. This permits high quality transverse, coronal
and sagittal images to be obtained directly. The absence of any
moving parts in MRI equipment promotes a high reliability. It is
believed that MRI or NMR imaging has a greater potential than CT
for the selective examination of tissue characteristics in view of
the fact that in CT, X-ray attenuation coefficients alone determine
image contrast, whereas at least four separate variables (T1, T2,
nuclear spin density and flow) may contribute to the NMR signal.
For example, it has been shown (Damadian, Science, Vol. 171, p.
1151, 1971) that the values of the T1 and T2 relaxation in tissues
are generally longer by about a factor of 2 in excised specimens of
neoplastic tissue compared with the host tissue.
By reason of its sensitivity to subtle physio-chemical differences
between organs and/or tissues, it is believed that MRI may be
capable of differentiating tissue types and in detecting diseases
which induce physicochemical changes that may not be detected by
X-ray or CT which are only sensitive to differences in the electron
density of tissue. The images obtainable by MRI techniques also
enable the physician to detect structures smaller than those
detectable by CT and thereby provide comparable or better spatial
resolution.
The use of perfluorocarbon compounds in various diagnostic imaging
technologies such as ultrasound, magnetic resonance, radiography
and computed tomography, has been set forth in an article by Robert
F. Mattrey in SPIE, Volume 626, Medicine, XIV/PACS IV (1986), pages
18-23 .
Magnetic resonance imaging of liver tumor and rats using
perfluorochemcial emulsions was reported in "In Vivo .sup.19 F NMR
Imaging of Liver, Tumor and Abcess in Rats", H. E. Longmaid III, et
al., INVESTIGATIVE RADIOLOGY, March -April 1985, Vol. 20, p.
141-144. The compounds utilized displayed multiple peak NMR
spectra.
Imaging of brain tumors with perfluorooctyl bromide has been
described in "Brain-Tumor Imaging Using Radiopaque
Perfluorocarbon", Nicholas J. Patronas, M.D., et al. JOURNAL OF
NEUROSURGERY, May 1983, Vol. 58, pp. 650-653.
Ultrasound imaging of organs has been enhanced by FLUOSOL-DA
(perfluorodecalin and perfluorotripropylamine) as reported in
"Perfluorochemicals as U. Contrast Agents for Tumor Imaging and
Hepatosplenography: Preliminary Clinical Results", Robert F.
Mattrey, M.D., et al., RADIOLOGY, May 1987, Vol. 163, No. 2, pp.
339-343.
In European published Patent Application 0 118 281, published Sep.
12, 1984, a technique for the detection of gas in an animal is set
forth using nuclear magnetic resonance techniques embodying various
fluorochemical agents. Among the fluorochemical agents there is
included perfluoro ether polymer (Fomblin Y/01).
In U.S. Pat. No. 4,523,039 the production of fluorocarbon ethers of
various structures is set forth wherein the resulting fluorocarbon
ether produces a noncyclic structure.
U.S. Pat. No. 4,570,004 describes a method of production and a
composition of matter including perfluoro 15-crown-5 ether. The
patent identifies that the crown ethers in general can be useful as
oxygen carriers and various biomedical products.
U.S. Pat. No. 4,639,364 discloses the use of various
fluorine-containing compounds for magnetic resonance imaging.
In parallel to the progress that has been made in the use of
Magnetic Resonance Imaging (MRI) as a clinical tool, in vivo NMR
spectroscopy (also called magnetic resonance spectroscopy, or MRS),
has been developed to probe human body chemistry noninvasively.
Efforts are being made to correlate the changes that are observed
in an NMR spectrum, such as the changes in chemical shifts and
areas of resonance peaks, to biochemical and metabolical states of
diseased organs. For example, knowledge of the concentrations of
high energy metabolites, such as adenosine triphosphate (ATP),
adenosine diphosphate (ADP), phosphocreatine (PC.sub.r), and
inorganic phosphate (Pi) derived from phosphorous (.sup.31 P) NMR,
can be used to determine whether or not a tissue is ischemic. It is
also known that tumors do affect the cell metabolism. By monitoring
the changes in the spectral features of the tumor, due to the
radiation treatments, it is possible to observe a patient's
progress without performing repeated biopsies. Progress has also
been made in monitoring the metabolic heterogeneity within a tumor
by in vivo human spectroscopy. The clinical applications are not
limited to these examples, but are mentioned in order to
demonstrate that in vivo human spectroscopy has potential future
clinical applications.
Today, the technology is still evolving. At this point, techniques
must be developed to define the volume of interest (VOI) to ensure
that the spectrum obtained is from the smallest region of interest
and not from the surrounding tissue. Therefore, the technological
challenge is to develop protocols which define controlled localized
diseased areas, which will definitely have an impact on the early
use of in vivo NMR spectroscopy as a routine clinical method for
diagnostic purposes. In addition to the need for accuracy of
localization, other desired features of an NMR spectrometer for
clinical applications are to provide: 1) the best sensitivity per
unit time, per unit volume; 2) the minimum of experimental time;
and 3) ease of operation.
The identified needs of a MRS perfluorchemical are for it to be: 1)
biocompatible and non-toxic; 2) having all fluorine atoms of
equivalent magnetic resonance; 3) have a T.sub.1 relaxation time
highly responsive to dissolved oxygen and not paramagnetic ions;
and 4) be cost effective.
Of the important nuclei in vivo spectroscopy, .sup.31 P remains the
most popular, since it offers a noninvasive, nondisruptive method
for providing information on the vital role of phosphorous in many
aspects of life processes. For example, .sup.31 P NMR signals
provide information on cellular energetics: phosphocreatine (PCr),
adenosine triphosphate (ATP), and inorganic phosphate (Pi). ATP has
been referred to as the "universal currency of free energy" in the
human body, mainly because of its widespread use as a carrier of
free energy within a cell. .sup.31 P NMR also provides information
concerning phospholipid syntheses and degradation, and also
synthesis of glycoproteins/glycolipids. It also permits the
measurement, invasively, of intracellular pH to indicate the
acid/alkaline state of the tissue. These measurements are useful in
understanding the state of health of tissues. Furthermore, the
increase or decrease of .sup.31 P resonance signal intensities of
an intact tissue is useful in probing the biochemical and
pathological aspects of diseased tissues. The changes in spectral
patterns can be used to diagnose and monitor the treatment for a
particular disease.
Parhamic and Fung established the characteristics of enhanced
.sup.19 F relaxation due to molecular oxygen and were one of the
first to foresee the possibility of using .sup.19 F NMR for in situ
determination of the amount of oxygen dissolved in body fluids,
organic or clinical studies. They investigated the current
important fluorochemicals such as cis- and trans-perfluorodecalin
and perfluorotributylamine. The logitudinal relaxation time
(1/T.sub.1) of each fluorine nuclei depended linearly on the
partial pressure of oxygen. The slopes of their plots were
different for each type of fluorine atom in the perfluorochemical.
They reasoned steric effects rather than specific binding of
molecular oxygen was the cause. P. Parhami and B. M. Fung,
Fluorine-19 Relaxation Study of Perfluoro Chemicals as Oxygen
Carriers, J. Physical Chemicals, 1983 pp 1928-1931.
Nunnally et al. showed that .sup.19 F-NMR spectra can be used to
monitor the rate of blood pool and extravascular space dilution of
a single bolus injection using emulsified fluorine species. Their
data were the first results of in vivo .sup.19 F-NMR studies of
perfusion and the determination of metabolism in specific organs
using .sup.19 F-NMR. R. L. Nunnally, R. M. Peshock, R. B. Rehr,
Fluorine-19 (.sup.19 F) NMR In Vivo. Potential For Flow And
Perfusion o Measurements, Proceeding of the Society of Magnetic
Resonance in Medicine, Second Annual Meeting Aug. 16-19, 1983 pg.
266.
In 1985 Reid et al. established that in a mixed fluorocarbon
emulsion the spin-lattice relaxation rates of the component .sup.19
F spectral lines were highly sensitive to oxygen concentration. R.
S. Reid, C. J. Koch, M. E. Castro, E. O. Trisben, D. J. P. Boisvert
and P. S. Allen. The Influence Of Oxygenation On The .sup.19 -Spin
Lattice Relaxation Rates Of Fluosol DA, Phys. Med. Biol. Vol 30 No.
7, pp. 677-686 1985.
Wyrwicz et al. have shown that by using .sup.19 F-NMR spectroscopy
they can detect small amounts (100-500 micromolar) of fluorinated
anesthetics in the brain of live animals during and after
anesthesia. A. M. Wyrwics, M. H. Pszenny, J. C. Schofield, R. E.
Gordon and P. A. Martin, Observations Of Fluorinated Anesthetics In
Rabbit Porain by .sup.19 F-NMR, Proceedings of the Society of
Magnetic Resonance in Medicine, Second Annual Meeting, Aug. 16-19,
183 pp 381-382.
Clark, et al. showed, under their conditions, the .sup.19 F-NMR
spectrum of perfluordecalin in emulsion, is not interfered with
from liver tissue. They found that perfluorochemical T.sub.1
relaxation times are insensitive to paramagnetic ions. L. C. Clark,
J. L. Ackerman, S. R. Thomas, R. W. Millard, R. E. Hoffman, R. G.
Pratt, H. Ragle-Cole, R. A. Kinsey and R. Janakiruman,
Perfluorinated Organic Liquids And Emulsions As Biocompatible NMR
Imaging Agents For .sup.19 F And Dissolved Oxygen, Adv. Exp. Med.
Biol. Vol. 180(6) pp 835-845, 1984.
The prior art, despite its suggestion for the use of magnetic
resonance spectroscopy for medical and biodiagnostic purposes and
the prior art's suggestion of various fluorine-containing compounds
for use as agents in nuclear magnetic resonance spectroscopy, has
failed to provide a particularly sensitive fluorine agent for
nuclear magnetic resonance spectroscopy which provides high signal
to noise ratios sufficient for detailed diagnosis of deep tissue
structures and unexpectedly high and diagnostically useful NMR
signal response to the presence to oxygen.
BRIEF SUMMARY OF THE INVENTION
The present invention is directed to a method for obtaining
fluorine magnetic resonance spectra from body organs or tissues by
administering to a mammal a fluorine-containing agent in a
sufficient amount to provide one or more fluorine magnetic
resonance spectra from said organs or tissues wherein the
improvement comprises using as said fluorine-containing agent
perfluoro 15-crown-5 ether.
Preferably, the perfluoro 15-crown-5 ether is administered in an
aqueous isotonic emulsion with a fluorochemical concentration range
of 5 to 25 wt %.
A particular embodiment of the present invention constitutes
administering the perfluoro 15-crown-5 ether in emulsion form into
body tissue for the purpose of quantifying the molecular oxygen
concentration by measuring the absolute spin-lattice relaxation
time (T.sub.1).
Another embodiment of the present invention is the measurement of
the rate of blood pool and extravascular space dilution by
following the change in absolute intensity of the single sharp
.sup.19 F-resonance line of perfluoro 15-crown-5 ether.
The perfluoro 15-crown-5 ether emulsion may be administered by a
technique of direct injection into a body part, a body compartment,
the bloodstream or by inhalation.
Alternatively, a method of the present invention can be performed
by administering the perfluoro 15-crown-5 ether to a mammal,
performing a biopsy of selected organ or body tissue and taking the
.sup.19 F spectrum of the biopsied tissue in vitro.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an NMR spectrum of perfluoro 15-crown-5 ether and a
perfluorobenzene standard.
FIG. 2 is an NMR spectrum of perfluoro 15-crown-5 ether taken alone
to shown the clean single response peak.
FIG. 3 is an NMR spectrum of perfluorodecalin taken alone to show
the multitude of response peaks which it gives as the state of the
art.
FIG. 4 is an NMR spectrum of an equimolar concentration of
perfluoro 15-crown-5 ether and perfluorodecalin showing that in
comparison to the amplitude of the former, the latter appears as
background noise response.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to the field of spectroscopy, in
contrast to the field of imaging which is disclosed in the present
inventors' U.S. Pat. No. 4,838,274, hereby incorporated by
reference herein in its entirety.
Fluorine atoms .sup.19 F give a clear nuclear magnetic resonance
signal and thus may function as suitable probes in nuclear magnetic
resonance imaging and spectroscopy when combined in a chemically
suitable form. The specific advantages flowing from the use of
.sup.19 F are:
(1) its low intrinsic concentration in soft tissues of the
body;
(2) its high nuclear magnetic resonance sensitivity, and
(3) a magnetogyric ratio which is close to that of hydrogen,
thereby making the observation of .sup.19 F compatible with
existing imaging and spectrographic devices.
However, the mere use of .sup.19 F in various compounds does not
provide the unexpected enhancement achieved by the present
invention wherein the use of perfluoro 15-crown-5 ether provides
the multiplied effect of 20 identically electronically and/or
magnetically situated fluorine atoms. This particular chemical
structure of fluorines provides a uniquely sharp signal when using
nuclear magnetic resonance imaging in a biocompatible
fluorine-containing agent. Perfluoro crown ethers have generally
been recognized as having utility in biomedical applications.
However, the present inventor has found that perfluoro 12-crown-4
ether is too volatile to be placed in the bloodstream of a mammal
because of its tendency to form embolisms. On the other hand,
perfluoro 18-crown-6 ether is too high in molecular weight for
biomedical application, and despite its emulsification in a
reasonably stable emulsion, when the agent is administered to a
mammal, the ether precipitates out as a solid and shows marked
toxicity.
Unexpectedly, perfluoro 15-crown-5 ether does not form embolisms
and does not precipitate out of emulsion when administered to a
mammal in an effective concentration sufficient for unexpectedly
high signal to noise response ratios in magnetic resonance
spectroscopy, particularly for diagnostic techniques for the
determination of oxygen in organs, particularly the spaces
surrounding and involved in tissue, tumors or cavities.
The unique location and association of fluorine atoms in perfluoro
crown ethers provides the single sharp resonance line of maximum
signal to noise ratio when used in magnetic resonance spectroscopy,
because of the magnetic equivalence of all the fluorine nuclei.
This provides the unique non-intrusive diagnostic capabilities of
perfluoro crown ethers as a diagnostic technique.
The perfluoro 15-crown-5 ether emulsion form is useful for nuclear
magnetic resonance diagnostic spectroscopy for diagnosis of tumors.
The perfluoro 15-crown-5 ether is capable of highlighting specific
biological dysfunctions. Additional diagnostic areas of interest
include cardiovascular blood transport, which can be observed for
site blockage, gastrointestinal constrictions which could be
outlined, lung capacity and tissue degeneration could be located
and tumor detection could be determined during early stages of
tumor development due to the heightened sensitivity of the specific
perfluoro crown ether.
The fluorine-containing agent of the present invention can be
administered within an organ, tissue, space, blood vessel or cavity
of a mammal. More specifically, the fluorine-containing agent of
the present invention can be administered by a process selected
from the group consisting of direct injection into a body part,
direct injection into a body cavity (thoracic, peritoneal), direct
injection into a body compartment (cerebrospinal fluid areas),
direct injection into a space (subarachnoid), direct injection into
a joint capsule, direct injection into the bloodstream, direct
injection into a growth, tumor, lump or swollen tissue area.
After the administration of the agent to the mammal, the desired
tissue can be biopsied and the biopsied and extracted tissue can be
analyzed to give an .sup.19 F-NMR spectrum.
The .sup.19 F-magnetic resonance spectrum of the fluorine agent of
the present invention can be used to monitor and quantify the
oxygen tension of the subject tissue, organ, space or cavity by
measurement of the spin-lattice (longitudinal) relaxation time
(1/T.sub.1).
The .sup.19 F-magnetic resonance spectrum of the fluorine agent of
the present invention can be used for the identification of an
isotense abnormal foci surrounded by normal tissue as a function of
the oxygen tension in and around the abnormal foci.
The .sup.19 F-magnetic resonance spectrum of the fluorine agent of
the present invention can also be used to monitor the rate of blood
pool and extravascular space dilution of a single bolus
injection.
Perfluoro 15-crown-5 ether was the only member of the perfluoro
crown ether class of materials identified in U.S. Pat. No.
4,570,004 (hereby incorporated herein by reference) to form a
stable aqueous emulsion at concentrations of 5 to 25 wt % in
sterile saline with nonionic surfactant systems and also provide
biocompatibility. This perfluoro crown ether was formulated into an
appropriate emulsion as set forth in the following examples.
EXAMPLE 1
An emulsion of perfluoro 15-crown-5 ether (PF15C-5E) was prepared
in sterile saline. One gram of perfluoro 15 crown-5 ether was
sonicated for 5 minutes at 20.degree. C. with 0.27 grams
supercritically extracted egg yolk lecithin in 4.5 grams of normal
saline.
EXAMPLE 2
In a typical control experiment, a normal, female Sprague-Dawley
rat weighing approximately 290 grams, was anesthetized with
ketamine hydrochloride. It was then injected directly into the
fourth ventrile with five (5) microliter aliquots of the PF15C-5E
emulsion (18 wt % fluorochemical) every five minutes until fifty
microliters was injected. The animal was then isolated. The animal
appeared normal and allowed to survive for 30 days. Gross
examination did not reveal any irregularity. It was concluded that
the fluorochemical emulsion was not toxic to the CNS (Central
Nervous System). No trace of the fluorochemical was found in the
animal after 30 days using capillary GC chromatography and an
electron capture detector.
EXAMPLE 3
In a typical experiment, a normal female Sprague-Dawley rat
weighing approximately 300 grams was anesthetized with ketamine
hydrochloride. After sedation, the animal was injected into the
fourth ventrile with five microliters of the perfluoro 15-crown-5
ether emulsion of Example 1 every five minutes until fifty
microliters had been injected. After one hour and forty-five
minutes an additional 0.13 cc of ketamine was injected to maintain
a constant level of sedation during .sup.19 F-NMR spectroscopy.
The .sup.19 F magnetic resonance spectrum was taken on a 1.4K Tesla
superconducting solenoid using a surface coil and time average
computer techniques to collect the NMR spectra from the rat's
brain. FIG. 1 gives the .sup.19 F magnetic resonance spectrum of
perfluoro 15-crown-5 ether in aqueous emulsion from the brain of
the rat. The reference signal of C.sub.6 F.sub.6 was obtained by
placing a capillary tube with C.sub.6 F.sub.6 under the surface
coil while the rat's brain was scanned.
EXAMPLE 4
In a typical experiment a Golden Hamster was injected into the
femoral vein with 1 ml of the perfluoro 15-crown-5 ether of Example
1. The animal, after being injected with 0.22ml of Ketaset, was
placed on its side on top of the NMR surface coil and a reference
standard capillary, C.sub.6 F.sub.6, placed underneath the animal.
Spectra were taken with a Phosphoenergetrics 30 cm horizontal bore
magnet operating at 2.2 Tesla. The resulting spectrum indicated
that the perfluoro 15-crown-5 ether could be located within the
animal's body near the location of the liver at the concentration
of injection. The animal was sacrificed and its organs excised.
Subsequent independent .sup.19 F magnetic resonance spectra were
taken of each organ. The perfluoro 15-crown-5 ether was found in
the spleen and liver confirming the in-vivo .sup.19 F NMR
analysis.
EXAMPLE 5
The .sup.19 F magnetic resonance relaxation time (1/T.sub.l) of
perfluoro 15-crown-5 ether was determined using a JEOL FX90Q
spectrometer. A perfluoro 15-crown-5 ether emulsion was prepared as
in Example 1. The stock emulsion was separated equally into three
(3) NMR tubes into which the oxygen concentration was established
to be zero, 18% and 100%, respectively. The NMR spectrum of each
sample was taken at 37.degree. C. The relaxation time, 1/T.sub.1,
of the NMR signal was found to change from 0.5 sec-1 to 0.87 sec-1
to 2.9 sec-1 with increasing partial pressure of oxygen. These in
vitro measurements of the spin lattice relaxation times (1/T.sub.1)
were found to compare with the in vivo measurement of 1/T.sub.1 in
hamsters, which were administered comparably oxygenated emulsions,
to give an estimate of the oxygen tension of the diagnosed tissue
of the hamsters with good experimentally correlated accuracy.
The advantage to using perfluoro 15-crown-5 ether is that the
compound's .sup.19 F-NMR spectrum consists of a single sharp line,
FIG. 2, while the present state of the art uses perfluorochemicals
that give multiple peaks, such as perfluorodecalin, FIG. 3. The
single peak of perfluoro 15-crown-5 ether represents 20
magnetically equivalent fluorines. Because of the 20 equivalent
fluorines, the relative sensitivity in the NMR of PF-15-crown-5
ether is five times greater than any other bio-acceptable
perfluoro-compound, see FIG. 4 for a comparison between
perfluoro-15-crown-5 ether and perfluorodecalin.
The disadvantage using compounds with more than one peak is
selecting which peak to monitor during the in vivo or in vitro
diagnostic analysis. If low field strength (<1.4 Tesla) magnets
are used the resolution of peaks is greatly diminished. This effect
causes the peaks of nearly equivalent nuclei (their chemical shift)
to coalesce into a broad unresolved peak. Because each equivalent
nuclei will respond to the presence of oxygen with a slightly
different relaxation time (1/T.sub.1), the final information is
comprised and of less diagnostic value. Higher field magnets
(>1.8 Tesla) can separate some of the chemical shifts. Larger
magnets are limited with today's technology to the size of the
magnet bore, thus reducing the size of the subject to be examined.
Perfluoro 15-crown-ether emulsions allow for a broader range of
application, contribute the greatest sensitivity per volume of
agent injected and directly yield the desired oxygen tension
information about the tissue or organ under evaluation without
applying mathametical conversions to the data.
The perfluoro-15-crown-5 ether emulsion is the ideal chemical
answer to a problem that has been addressed with mechanical and
mathematical approaches for the past 10 years, that is, how to
isolate a single .sup.19 F-NMR resonance for in vivo diagnostic
applications.
The present invention has been set forth with emphasis of a
particular preferred embodiment. However, the scope of the present
invention should be ascertained from the claims which follow.
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