U.S. patent application number 11/973926 was filed with the patent office on 2008-09-04 for method for monitoring blood flow and metabolic uptake in tissue with radiolabeled alkanoic acid.
This patent application is currently assigned to The General Hospital Corporation. Invention is credited to David R. Elmaleh, Alan J. Fischman, Timothy M. Shoup.
Application Number | 20080214851 11/973926 |
Document ID | / |
Family ID | 33310796 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080214851 |
Kind Code |
A1 |
Elmaleh; David R. ; et
al. |
September 4, 2008 |
Method for monitoring blood flow and metabolic uptake in tissue
with radiolabeled alkanoic acid
Abstract
The present invention relates to novel modified fatty acid
analogs, where a positron or gamma-emitting label is placed at a
position on a fatty acid backbone and an organic substituent is
substituted at 2,3; 3,4; 4,5; 5,6 and other sequence positions of a
fatty acid backbone. Theses novel fatty acid analogs are designed
to enter the tissues of interest by the same long chain fatty acid
carrier mechanism as natural fatty acids, however, functional
substituents in the 2,3; 3,4; 4,5; 5,6 and other sequence
positions, block the catabolic pathway, thus trapping these analogs
in a virtually unmodified form in the tissues of interest.
Inventors: |
Elmaleh; David R.; (Boston,
MA) ; Fischman; Alan J.; (Boston, MA) ; Shoup;
Timothy M.; (Boston, MA) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
The General Hospital
Corporation
Boston
MA
|
Family ID: |
33310796 |
Appl. No.: |
11/973926 |
Filed: |
October 11, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10827054 |
Apr 19, 2004 |
|
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|
11973926 |
|
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60463574 |
Apr 17, 2003 |
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Current U.S.
Class: |
554/124 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 51/0402 20130101 |
Class at
Publication: |
554/124 |
International
Class: |
C07C 51/00 20060101
C07C051/00 |
Claims
1-129. (canceled)
130. A method of synthesizing a radioactively labeled analog of a
fatty acid having the formula:
R--(CH.sub.2).sub.nZ-(CH.sub.2).sub.mCO.sub.2H, wherein n is 8-22,
m is 0-10, r is a CH.sub.3, aryl or a heterocyclic group, and Z is
a cyclic or heterocyclic organic substituent which causes said
analog to be metabolically trapped in a tissue according to claim
1, comprising the steps of: a. synthesizing a mono-protected
primary alcohol from a starting compound; b. adding a cyclic or
heterocyclic organic substituent to the mono-protected primary
alcohol to form a cyclic mono-protected primary alcohol; and c.
treating the cyclic mono-protected primary alcohol to form the
radioactively labeled fatty acid analog.
131. The method of claim 130, wherein the starting compound
comprises a carbon backbone that is saturated.
132. The method of claim 130, wherein the starting compound
comprises a carbon backbone that is unsaturated.
133. The method of claim 130, wherein the starting compound
comprises a terminal phenyl group.
134. The method of claim 130, wherein the starting compound is a
cyclic primary alcohol.
135. The method of claim 130, wherein the cyclic organic
substituent is a cyclic alkane.
136. The method of claim 135, wherein the cyclic alkane is selected
from the group consisting of cyclopropyl, cyclobutyl and
cyclopentyl.
137. The method of claim 130, wherein said heterocyclic organic
substituent comprises a 3-5-membered heterocyclic ring
structure.
138. The method of claim 130, further comprising adding a
radioactive label that is bonded to a carbon atom of the
analog.
139. The method of claim 138, wherein the radioactive label is
selected from the group consisting of: .sup.18F, .sup.123I,
.sup.131I, .sup.34mCl, .sup.75Br, .sup.76Br or .sup.77Br.
140. A kit for administration of a radioactively labeled analog of
a fatty acid, comprising an analog of a fatty acid synthesized
according to claim 130, a radioactive isotope, a pharmaceutically
acceptable carrier, and optionally instructions for preparing the
radioactively labeled analog or use thereof.
141. The kit of claim 140, wherein the radioactive isotope is
selected from the group consisting of: .sup.18F, .sup.123I,
.sup.131I, .sup.34mCl, .sup.75Br, .sup.76Br or .sup.77Br.
142-146. (canceled)
Description
REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCE
[0001] This application claims priority to U.S. Application Ser.
No. 60/463,574, filed on Apr. 17, 2003.
[0002] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the U.S.
and foreign applications or patents corresponding to and/or
claiming priority from any of these applications and patents, and
each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference. More generally, documents of references are
cited in this text, either in a Reference List before the claims,
or in the text itself; and, each of these documents or references
("herein-cited references"), as well as each document or reference
cited in each of the herein-cited references (including any
manufacturer's specifications, instructions, etc.), is hereby
expressly incorporated herein by reference. Documents incorporated
by reference into this text may be employed in the practice of the
invention. This application makes reference to U.S. Pat. No.
4,524,059, issued on Jun. 18, 1985, the contents of which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to novel radiolabeled fatty
acid analogs having a cyclic organic substituent, where a positron
or gamma-emitting label is placed at a position on a fatty acid
backbone and an organic substituent is substituted at the 2,3; 3,4;
4,5; 5,6 and other sequence positions of a fatty acid backbone.
These novel fatty acid analogs are designed to enter the tissues of
interest by the same long chain fatty acid carrier mechanism as
natural fatty acids, however, functional substituents in the 2,3;
3,4; 4,5; 5,6 and other sequence positions, block the catabolic
pathway, thus trapping these analogs in a virtually unmodified form
in the tissues of interest.
BACKGROUND OF THE INVENTION
[0004] Many imaging modalities have been developed for non-invasive
evaluation of heart disease. Radiotracers that can assess
myocardial perfusion and heart metabolism are useful for clinical
evaluation of ischemic heart disease and cardiomyopathies. Fatty
acids (FAs) are the principal substrate for the production of
adenosine triphosphate (ATP) in the myocardium under aerobic
conditions. Nearly 70% of myocardial energy results from metabolism
of fat in the basal state, with the remainder of the myocardial
energy requirements supplied by glucose (15%), lactate and pyruvate
(12%) and amino acids (5%). Therefore, FAs and modified fatty acids
(MFAs) have been proposed for imaging the heart.
[0005] Uptake of free FAs by the myocardium occurs at an extraction
percentage of 40% to 60% of blood content, which is proportional to
perfusion. Transported to the heart as nonesterified FAs, as
triglycerides in very low-density lipoprotein particles or in
chylomicrons, or bound to serum albumin, they pass along
concentration gradients to the interstitium. Under these
conditions, FAs supply as much as 70% of oxidatively metabolized
substrate. The extraction of free FAs by the myocyte is regulated
by several variables including FA chain length, the availability of
other metabolic substrates, circulating levels of hormones, cardiac
workload, and the presence or absence of ischemia.
[0006] MFAs behave like native FAs up to the .beta.-oxidation step
in the mitochondria, where it is sequestered for a long period of
time (Livni, E. et al. 1990 Lipids 25: 238-40). Highly simplified,
the fate of an FA may be described as follows: FA passes from
capillary blood into the interstitial space. It may "back-diffuse"
to the vascular space or pass through the interstitial space, where
it may become activated as acyl-coenzymeA (CoA). The activated FA
can then be esterified to form triglycerides, incorporated into
phospholipids or carried into the mitochondria and oxidized. The
activation of FA to acyl-CoA requires energy and is believed to be
essentially irreversible in vivo. Since acyl-CoA cannot escape
through the cell membrane, it becomes trapped in the cell. However,
the formation of triglycerides is not irreversible and these can be
broken down into the constituent FA and glycerol, adding to the FA
pool.
[0007] As described above, there is an intimate relationship
between FA metabolism and myocardial integrity. As a result of
their high rate of utilization, labeled FAs in conjunction with
suitable detection techniques can provide a means of quantifying in
vivo regional myocardial metabolism. Two approaches can be employed
to quantify the utilization of substrates in vivo. The first
involves the use of radiolabeled, positron emitting physiologic
substrates. This approach has been used to assess glucose and FA
metabolism in the heart with carbon 11 (.sup.11C) palmitate (PA) in
rabbit (Raichle, M. E. et al. (1978) Science 199: 986-987). It has
been demonstrated that after brief intervals of ischemia, PA
extraction fell markedly, even when alterations in cardiac function
were reversible (Klein M. S. et al. (1979) Am. J. Physiol. 237:
H51-H58; Goldstein R. A. et al. (1980) J. Nucl. Med. 21: 342-348;
Weiss, E. S. et al. (1969) Circulation 19: 25-32; Fox, K. A. et al.
(1975) Circ. Res. 57: 232-243; Hoffman, E. J. et al. (1977) J.
Nucl. Med. 18: 57-61; Ter-Pogossian, M. M. et al. (1980)
Circulation 61: 242-255; Weiss E. S. et al. (1977) Circulation 55:
66-73; Schon, H. R. et al. (1982) Am. Heart J 103: 532-547; Schon,
H. R. et al. (1982) Am. Heart J. 103: 548-561; Schelbert, H. R. et
al. (1983) Am. Heart J. 105: 492-504; Schelbert, H. R. et al.
(1983) Am. Heart J. 106: 736-50; Sobel, B. E. (1982) Am. Heart J.
103: 673-681; Schelbert, H. R. et al. (1983) Am. Heart J. 105:
522-526; Schelbert, H. R. (1985) Circulation 72: TV122-133; Sobel,
B. E. (1985) Circulation 72:IV22-30; Rosamond, T. L. et al (1987)
J. Nucl. Med. 28:1322-1329; Grover-McKay, M. et al. (1986)
Circulation 74: 281-292; Schelbert, H. R. et al (1986) Am. Heart J.
111: 1055-1064; Jaffe, A. S. et al (1987) Int. J. Cardiol. 15:
77-89; Knabb, R. M. et al (1987) J. Nucl. Med. 28:1563-1570; Myars,
D. W. et al. (1987) Am. J. Physiol. 253: 107-114). In addition,
zones of persistently decreased flow also showed decreased PA
extraction (Schelbert, H. R. (1985) Circulation 72: TV122-133).
[0008] Other studies using .sup.11C-PA provide the experimental
basis for studying regional myocardial FA distribution. The
application of physiologic radiolabeled FAs to in vivo
quantification of regional myocardial metabolic rates suffers from
several drawbacks. First, the use of .sup.11C-FAs labeled with
.sup.11C on the carboxyl group is subject to loss of labeling
during the first round of .beta.-oxidation. Studies employing
direct intracoronary administration of .sup.11CO.sub.2 and direct
myocardial monitoring demonstrate the evolution of .sup.11CO.sub.2
within 30 seconds and a 50% clearance within 2-8 minutes. Secondly,
the rapid washout of the radiolabel due to .beta.-oxidation and
short sequential imaging periods imposes limitations on counting
statistics. Third, in the myocardial cell, FAs are distributed
among different pools: free FAs, triglycerides, phospholipids,
diglycerides, and monoglycerides. It is unclear whether the rate of
FA oxidation is proportional to the rate of triglyceride
hydrolysis. After the initial clearance of radioactivity from the
blood, there is a rise in activity due to the release of
radiolabeled metabolites from the liver. This makes the completion
of labeled FA detection necessary before the rise occurs. Other
studies in dogs using .sup.11C-PA showed that during ischemia,
quantitation is limited due to the complexity of its metabolic fate
(Schelbert et al., 1983 Am. Heart J 106:736-50). .sup.11C acetate
has been proposed as an alternative due to its simpler metabolic
fate.
[0009] The second approach involves the use of "analog tracers"
that enter a known metabolic pathway. However, because of their
unique chemical structure, metabolism of these tracers stop at a
certain stage, leaving the radiolabel trapped in the cell in a
known form. This concept has been applied to the study of glucose
metabolism using glucose analogs such as
1-[.sup.11C]-2-deoxyglucose (2DG) and
2-[.sup.18F]-fluorodeoxyglucose (2FDG). The principle of metabolic
trapping has been used successfully with 2FDG to measure in vivo
regional glucose metabolic rates in humans. Investigations of the
use of 2FDG for measuring myocardial glucose metabolism have been
conducted. Similarly, FAs have also been widely used to measure
metabolic activity in the myocardium. A major drawback to the use
of FAs is the quick washout rates, as alluded above. FAs tend to
wash out very quickly due to .beta.-oxidation, depending on the
position of the radionuclide. Subsequently, the radiolabeled FA or
metabolites can then accumulate in tissues other than the region of
interest, primarily liver and lung. In radiohalogenated aliphatic
fatty acids, such accumulation occurs frequently with 1231, which
migrates and is stored in the thyroid gland, and .sup.18F, which is
stored in bone.
[0010] Evans and coworkers radiolabeled straight-chain FAs by
saturation of the double carbon bond of oleic acid with .sup.131I
and found that although photoscans of the canine heart were
produced, the low specific activity of the final product precluded
its clinical use (Evans, J. et al (1965) Circ. Res. 16: 1-10;
reviewed in Corbett, J. R., (1999) Semin. Nucl. Med. 29(3):
1999-2006). Since then, there have been many MFAs developed for
cardiac imaging. Poe and coworkers showed that
[.sup.123I]-hexadecanoic acid (IHXA) and [.sup.123I]-heptadecanoic
acid (IHDA) were indicators of myocardial perfusion in experimental
canine models and demonstrated clearance rates similar to that of
.sup.11C-PA (Poe, N. et al, (1976) J. Nucl. Med. 16; 17-21; Poe, N.
et al, (1977) Radiology 124: 419-424). All subjects with prior
myocardial infarcts showed decreased regional tracer uptake.
Machulla et al reported that the .omega.-terminal labeled FAs were
more efficiently extracted than analogs labeled in the
.alpha.-position, and that IHDA had the highest uptake (Machulla,
H. et al (1978) J. Nucl. Med. 19: 298-302).
[0011] Although IHDA and 1.times.A have potential as myocardial
perfusion agents, their ability to access myocardial metabolism in
patients has been questioned (Freundlieb, C. et al, (1980 J. Nucl.
Med. 21(11): 1043-50; Visser, F. C. et al, (1985) Circulation
72(3): 565-71). The clinical utility of radiolabeled iodoalkyl FAs
appears limited by: 1) the rapid appearance of free radioiodine,
requiring special correction procedures to differentiate between
myocardial and blood pool activity; 2) short elimination
half-lives, making them unattractive agents for single photon
imaging; and 3) data suggesting that the elimination rate may not
reflect .beta.-oxidation but rather de-iodination and
back-diffusion of the tracer across the membrane. Further,
protocols and algorithms developed for planar imaging are not
applicable to single photon imaging (SPECT), effectively
eliminating it as a potential imaging modality for the measurement
of metabolic parameters with these radiotracers. Imaging
difficulties associated with de-iodination of IHDA and IHXA
ultimately resulted in the development of the branched FAs.
[0012] The molecular structure of FAs can be modified to attenuate
myocardial metabolism, prolong cardiac retention, and avoid washout
effects. To prevent rapid de-iodination of alkyl FAs and promote
stabilization of the iodine radiolabel, 15-(p-iodophenyl)
pentadecanoic acid (IPPA) was developed as an alternative
(Machulla, H. et al. (1980) Eur. J. Nucl. Med. 5: 171-173). The
.sup.123I-label attached to a terminal phenyl ring in either the
ortho or para position is stabilized against de-iodination.
[123I]-IPPA has kinetics similar to the physiological substrate
.sup.11C-PA in perfused rat hearts (Reske, S. et al, (1984) J.
Nucl. Med. 25:1335-1342). The uptake of IPPA is related to
perfusion, and IPPA generally follows the normal metabolic pathway
for .beta.-oxidation (Caldwell, J. et al, (1990) J. Nuc. Med. 31:
99-105). Iodobenzoic acid and its metabolite iodohippurate are the
products of IPPA oxidation, which are rapidly excreted by the
kidneys with the iodine moiety still attached, preventing buildup
of free radioiodine.
[0013] Studies using myocardial biopsy specimens have shown rapid
extraction by normal myocardium, with biexponential clearance,
including a fast component t.sub.1/2 of 3.5 minutes (flow), a slow
component t.sub.1/2=130 minutes (metabolism), and a blood clearance
t.sub.1/2=5 minutes (elimination) (Chien, K. et al (1983) Am. J.
Physiol. 245: H693-H697). Compared with alkyl straight-chain FAs,
IPPA has the advantages of rapid myocardial uptake, iodine
stabilization, and rapid clearance of metabolites from the body.
While IPPA was a significant improvement over the straight chain
FAs, providing excellent image quality, and permitting SPECT image
acquisition and quantification with estimates of metabolic rates,
the rate of IPPA metabolism and clearance was still relatively fast
for SPECT imaging. Thus, an effort was made to develop radiolabeled
FA analogs with attenuated oxidative metabolism.
[0014] Methyl branching was introduced to slow myocardial clearance
and improve quantitative image accuracy (Livni, E. et al. 1982 J.
Nucl. Med. 23: 169-75; Elmaleh, D. R. et al. 1981. J. Nucl. Med.
22: 994-9; Elmaleh, D. R. et al. 1983 Int. J. Nucl. Med. Biol.
10:181-7; Goodman, M. M. et al. 1984 J. Org. Chem. 49: 2322-5;
Livni, E. et al. 1985. Eur. Heart J. 6 (Suppl B): 85-9; Bianco, J.
A. et al. Eur. J. Nucl. Med. 12: 120-4). The addition of methyl
group(s) at the 3-carbon position blocks .beta.-oxidation by
preventing formation of the 3-carboxy-intermediate
(.beta.-ketoacyl-ScoA) via the dehydrogenation of the 5-L-hydroxy
ScoA intermediate. Two iodine-labeled MFAs that provide prolonged
myocardial retention are
15-(p-iodophenyl)-3-(R,S)-methyl-pentadecanoic acid (BMIPP) and
15-(p-iodophenyl)-3,3-dimethyl-pentadecanoic acid (DMIPP). The
kinetics and subcellular distribution of these methyl-branched FAs
have been evaluated, with DMIPP demonstrating the greatest
myocardial retention times, with no significant metabolism (Knapp,
F. et al, (1986) Eur. J. Nucl. Med. 12: S39-S44; Ambrose, K. et al,
(1987) Eur. J. Nucl. Med. 12: 486-491). However, DMIPP demonstrates
very slow myocardial clearance (6-7 hours), limiting its usefulness
for certain applications. Additionally, DMIPP has been detected in
exogenous tissues, such as the liver (Sloof et al, (1997) Nucl.
Med. Commun. 18(11): 1065-70). BMIPP also accumulates in the liver,
but to a lesser extent than DMIPP.
[0015] BMIPP is currently the most widely used radiolabeled MFA for
cardiac imaging. BMIPP has prolonged myocardial retention (30-45
minutes) and undergoes .beta.-oxidation in the myocyte after the
initial .alpha.-oxidation and oxidative decarboxylation, producing
.alpha.-hydroxy-BMIPP as an intermediate (Yamamichi, Y. et al,
(1995) J. Nucl. Med. 36: 1042-1050). After loss of propionic acid,
further degradation proceeds through successive cycles of
.beta.-oxidation to the end product, (p-iodophenyl)-acetic acid.
Additionally, it has been shown that initial distribution of BMIPP
in the first several minutes after injection is comparable to that
of perfusion tracers like .sup.201Thallium and .sup.99m technetium
compounds. Thus, it can be argued that the use of BMIPP alone
imaged early and late after injection is all that is required to
evaluate myocardial viability with a high degree of accuracy.
Likewise, numerous groups have accomplished work on FAs as
potential markers for blood flow and their collective data
determined that a single injection of certain FAs produces images
that are similar to those produced by .sup.201TI, .sup.11C-PA, or
potassium (van der Wall, E. E. et al, (1980) Eur. J. Nucl. Med.
5(5): 401-5; Kairento, A. L. et al, (1988) Int. J. Rad. Appl.
Instrum. B. 15(3): 333-8; Kobayashi, H. et al, (1997) J. Nucl. Med.
39(7): 1117-22; Kawamoto, M. et al, (1994) J. Nucl. Cardiol. 1(6):
522-8). Thus, a single injection limits the amount of radioactivity
exposure to the patient, as well as being cost-effective. While
BMIPP is a widely used FA for both flow and metabolism, there is a
need in the art for MFAs that have longer myocardial retention
without appreciable metabolism and migration of the radiolabel to
other unwanted areas. BMIPP and its metabolites have been detected
at significant levels in lung tissue (Sloof et al, (1997) Nucl.
Med. Commun. 18(11): 1065-70).
[0016] While there are a plurality of MFAs that are suitable for
imaging, many either undergo significant .beta.-oxidation,
resulting in production of radiolabeled metabolites that can
accumulate in other tissues, or fail to be efficiently transported
into the myocardium, resulting in back-diffusion and
re-esterification into the triglyceride pool. Thus, there is a need
in the art for a radiolabeled MFA analog that is transported to the
tissue of interest by endogenous, physiological means, but fails to
undergo .beta.-oxidation due to the presence of branched organic
substituents. This would allow the MFA to be retained in a
virtually unmodified form for a sufficient amount of time in the
tissue of interest (i.e. cardiac tissue) to be detected by
conventional means. Further, there is a need in the art for a
radiolabeled MFA that shares the aforementioned characteristics and
is retained without significant migration to unwanted areas of the
organism.
SUMMARY OF THE INVENTION
[0017] Alterations in FA uptake are considered to be sensitive
markers of ischemia and myocardial damage. Radiolabeled FAs that
initially display myocardial blood flow and eventually show
adequate myocardial retention associated with metabolic activity
are attractive candidates for clinical evaluation of regional
discrepancies in heart blood flow and FA metabolism, which occur in
ischemic heart disease and cardiomyopathies.
[0018] Therefore, the present invention relates to novel
radiolabeled FA analogs having a cyclic organic substituent, where
a positron or gamma-emitting label is placed at a position on an FA
backbone and an organic substituent is substituted at the 2,3; 3,4;
4,5; 5,6 and other sequence positions of an FA backbone. These
novel FA analogs are designed to enter the tissues of interest by
the same long chain FA carrier mechanism as natural FAs, however,
functional substituents in the 2,3; 3,4; 4,5; 5,6 and other
sequence positions, block the catabolic pathway, thus trapping
these analogs in the tissues of interest.
[0019] Accordingly, in one aspect of the present invention, a
radioactively labeled analog of a fatty acid that is taken up by
mammalian tissue is provided, comprising the formula:
R--(CH.sub.2).sub.n-Z-(CH.sub.2).sub.mCO.sub.2H,
wherein n is 8-22, m is 0-10, R is a CH.sub.3, aryl or a
heterocyclic group, and Z is a cyclic or heterocyclic organic
substituent which causes said analog to be metabolically trapped in
said tissue.
[0020] The term "aryl" in the context of this application may
comprise, but is not limited to, a 5-, 6- or 7-membered ring.
Further, the term "cyclic" can refer to cyclic alkanes such as
cyclopropyl, cyclobutyl, and cyclopentyl, but is not so limited.
Similarly, "heterocylic" can refer to any 3 to 5-membered ring
structure that can comprise, for example, nitrogen, sulfur, or
oxygen atoms.
[0021] In another aspect, a radioactively labeled analog of a fatty
acid that is taken up by mammalian tissue is provided, comprising
the formula:
##STR00001##
[0022] wherein D is CH.sub.2 or CH.sub.2CH.sub.2, E is CH.sub.2 or
CH.sub.2CH.sub.2, m is 0-10, n is 8-14 and R is a CH.sub.3, aryl or
a heterocyclic group, wherein cyclic organic substituent
-CDCE-causes said analog to be metabolically trapped in said
tissue.
[0023] Another aspect of the present invention provides a
radioactively labeled analog of a fatty acid, comprising the
formula:
##STR00002##
wherein A can be (CH.sub.2).sub.X, O, or S; X is 1, 2, 3, and 4;
cis and trans isomers R,R and S,S and their racemic forms; m is
0-10; n is 14-8; R is .sup.18F-phenyl, or 123I-phenyl, and wherein
the cyclic or heterocyclic organic substituent --CH-A-CH-- causes
said analog to be metabolically trapped in said tissue.
[0024] In another aspect of the present invention, a radioactively
labeled analog of a fatty acid that is taken up by mammalian tissue
is provided, comprising the formula:
##STR00003##
wherein A is (CH.sub.2).sub.y, O, or S; y is 1, 2, 3, 4; cis and
trans isomers R,R, and S,S and their racemic forms; m is 0-10; n is
14-8; p is 0-6; R is CH.sub.3; and X is a radioactive label.
[0025] The present invention also provides a radioactively labeled
analog of a fatty acid that is taken up by mammalian tissue,
comprising the formula:
##STR00004##
wherein X can be H, .sup.18F, .sup.123I, .sup.131I, .sup.34mCl,
.sup.75Br, .sup.76Br, .sup.77Br, alkyl and heteroalkyls thereof; Y
can be H, .sup.18F, .sup.123I, .sup.131I, .sup.34mCl, .sup.75Br,
.sup.76Br, .sup.77Br, alkyl and heteroalkyls thereof; A is
(CH.sub.2).sub.z, O or S; Z is 1-4, m is 0-10, n is 8-14, p is 0-6,
R is CH.sub.3, aryl or heterocyclic, and wherein the cyclic or
heterocyclic organic substituent --CH-A-CH-- causes said analog to
be metabolically trapped in said tissue.
[0026] Another aspect of the present invention provides a
radioactively labeled analog of a fatty acid that is taken up by
mammalian tissue, comprising the formula:
##STR00005##
wherein A is (CH.sub.2).sub.z, O or S, y is 1-4, m is 1-10, n is
8-14, p is 0-6, R is CH.sub.3, aryl or heterocyclic, X can be
.sup.18F, .sup.123I, .sup.131I, .sup.34mCl, .sup.75Br, .sup.76Br or
.sup.77Br; and wherein the cyclic or heterocyclic organic
substituent --CH-A-CH-- causes said analog to be metabolically
trapped in said tissue.
[0027] Preferably, the novel fatty acid has a cyclic organic
substituent labeled with .sup.18F, .sup.123I, .sup.131I,
.sup.34mCl, .sup.75Br, .sup.76Br and .sup.77Br.
[0028] In another embodiment, the novel fatty acid having a cyclic
organic substituent is saturated.
[0029] Another embodiment of the present invention describes the
novel fatty acid having a cyclic organic substituent that contains
one or more double bonds.
[0030] In one embodiment, the organic substituent is a 3-to-5
membered cyclic structure.
[0031] Another aspect of the present invention provides a method of
measuring blood flow in a subject, comprising the following steps:
[0032] a) localizing a detectable amount of a FA composition of the
invention to a tissue of interest; [0033] b) detecting a signal
from said FA composition in a tissue of interest within about 1
minutes and about 5 minutes after administration; [0034] c) imaging
a tissue of interest and; [0035] d) determining the rate of blood
flow in a tissue of interest.
[0036] In another aspect, a method for measuring metabolism in a
subject comprises the following steps: [0037] a) localizing a
detectable amount of a FA composition of the invention to a tissue
of interest; [0038] b) detecting a signal from said FA composition
in a tissue of interest within about 30 minutes and about 120
minutes after administration; [0039] c) imaging a tissue of
interest and; [0040] d) determining the rate of metabolism in a
tissue of interest.
[0041] Another aspect of the present invention provides a method
for retaining a fatty acid composition of the invention in a tissue
of interest, comprising the steps of: [0042] a. localizing a
detectable amount of the composition to the tissue; [0043] b.
retaining the composition, or a metabolic derivative thereof, in
the tissue by reducing transport and back-diffusion of the
composition; and [0044] c. detecting the composition or the
metabolic derivative in the tissue.
[0045] The present invention further provides a method for
retaining a fatty acid composition of the invention in a tissue of
interest, comprising the steps of: [0046] a. localizing a
detectable amount of the composition to the tissue; [0047] b.
retaining the composition, or a metabolic derivative thereof, in
the tissue by reducing dehydrogenation of the composition; and
[0048] c. detecting the composition or the metabolic derivative in
the tissue.
[0049] Another aspect provides a method for retaining a fatty acid
composition of the invention in a tissue of interest, comprising
the steps of: [0050] a. localizing a detectable amount of the
composition to the tissue; [0051] b. retaining the composition, or
a metabolic derivative thereof, in the tissue by reducing
hydroxylation of the composition; and [0052] c. detecting the
composition or the metabolic derivative in the tissue.
[0053] The present invention further provides a method for
retaining a fatty acid composition of the invention in a tissue of
interest, comprising the steps of: [0054] a. localizing a
detectable amount of the composition to the tissue; [0055] b.
retaining the composition, or a metabolic derivative thereof, in
the tissue by reducing ketoacyl formation of the composition; and
[0056] c. detecting the composition or the metabolic derivative in
the tissue.
[0057] Yet another aspect of the present invention provides a
method for retaining a fatty acid composition of the invention in a
tissue of interest, comprising the steps of: [0058] a. localizing a
detectable amount of the composition to the tissue; [0059] b.
retaining the composition, or a metabolic derivative thereof, in
the tissue by reducing ketoacetyl elimination of the composition;
and [0060] c. detecting the composition or the metabolic derivative
in the tissue.
[0061] In one embodiment, the tissue of interest is heart
tissue.
[0062] In another embodiment, the tissue of interest is liver
tissue.
[0063] The present invention preferably describes tumor tissue as
the tissue of interest.
[0064] In one embodiment, the tissue is diseased.
[0065] In another embodiment, the tissue is healthy.
[0066] Preferably, the tissue can be subjected to exercise-induced
stress or pharmacologically induced stress.
[0067] Another aspect of the present invention provides a method of
synthesizing a fatty acid composition of the invention, comprising
the steps of: [0068] a) synthesizing a mono-protected primary
alcohol from a starting compound; [0069] b) adding a cyclic or
heterocyclic organic substituent to the mono-protected primary
alcohol to form a cyclic mono-protected primary alcohol; and [0070]
c) treating the cyclic mono-protected primary alcohol to form the
fatty acid analog.
[0071] One embodiment of the present invention describes the
starting compound comprising a carbon backbone that is
saturated.
[0072] In another embodiment, the starting compound comprises a
carbon backbone that unsaturated.
[0073] Another embodiment describes the starting compound
comprising a terminal phenyl group.
[0074] In another embodiment, the starting compound is a cyclic
primary alcohol.
[0075] One embodiment of the present invention describes a cyclic
alkane as the cyclic organic substituent.
[0076] Another embodiment describes the heterocyclic organic
substituent as a 3-5-membered heterocyclic ring structure.
[0077] In another embodiment, the method further comprises adding a
radioactive label that is bonded to a carbon atom of the
analog.
[0078] The present invention further provides a kit for
administration of a radioactively labeled analog of a fatty acid,
comprising an analog of a fatty acid synthesized according to the
methods of the invention, a radioactive isotope, a pharmaceutically
acceptable carrier, and optionally instructions for preparing the
radioactively labeled analog or use thereof.
[0079] Preferably, the radioactive isotope is selected from the
group consisting of .sup.18F, .sup.123I, .sup.113I, .sup.34mCl,
.sup.75Br, .sup.76Br aid .sup.77Br.
[0080] Throughout this specification and claims, the word
"comprise" or variations such as "comprises" or "comprising" will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers.
[0081] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0082] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
Figures, incorporated herein by reference.
[0083] FIG. 1 is a schematic overview of the synthesis of
[.sup.15F]-9-fluoro-3,4-cyclopropyl-heptadecanoic acid.
[0084] FIG. 2 depicts the general formula of a saturated fatty acid
comprising a substituted radiolabel directly on the fatty acid
backbone. This figure of a fatty acid variant comprises a
generalized structure similar to the compound in FIG. 1.
[0085] FIG. 3 depicts [.sup.18F]-FCPHA activity in the heart of a
monkey as a function of time.
[0086] FIG. 4 shows heart images of a pig at 2-8 minutes after
intravenous injection of 18 mCi of [.sup.13N]-ammonia.
[0087] FIG. 5 shows heart images of the same pig in FIG. 5, at 2-8
minutes after intravenous injection of 19 mCi [.sup.18F]-FCPHA.
[0088] FIG. 6 is a comparison of [.sup.13N]-ammonia (right) and
[.sup.18F]FCPHA (left) images obtained from a pig at 2-8 minutes
after tracer injection.
[0089] FIG. 7 is an image collected by positron emission tomography
of the biodistribution of [.sup.18F]-FCPHA in the left and right
ventricles of a monkey heart.
[0090] FIG. 8 shows transverse heart level slices of a monkey 60
minutes after administration of [.sup.18F]-FCPHA.
[0091] FIG. 9 depicts the general formula of a terminally labeled
straight chain fatty acid comprising a substituent designated by
`A` and a phenyl moiety comprising a substituted radiolabel.
[0092] FIG. 10 is a schematic overview of the synthesis of
endo-[.sup.18F] fluoro- or [.sup.123I]
iodo-3,4-cyclopropylheptadecanoic acid.
[0093] FIG. 11 depicts the general formula of a fatty acid
comprising an endo-vinyl group. This endo-vinyl group can comprise
a substituted radiolabel at substituents `X` or `Y`.
[0094] FIG. 12 is a schematic overview of the synthesis of
exo-[.sup.18F] fluoro or [.sup.123I]
iodo-3,4-cyclopropylheptadecanoic acid.
[0095] FIG. 13 depicts the general formula of a fatty acid
comprising an exo-vinyl group. This exo-vinyl group can comprise a
substituted radiolabel at substituents `X` or `Y`.
[0096] FIG. 14 shows the partial synthesis of a portion of a
modified fatty acid comprising a substituent that can be a four- to
five-membered ring structure.
[0097] FIG. 15 shows the general formula of a modified fatty acid
comprising a substituent that can be a four- to five-membered ring
structure. This fatty acid variant can comprise a combination of
features described in FIGS. 2, 4, 11, and 13.
DETAILED DESCRIPTION OF THE INVENTION
[0098] In accordance with the instant invention, novel radiolabeled
fatty acid compositions having a cyclic organic substituent can be
administered to an animal, including a human, to determine both
blood flow to the organ and metabolism by an organ of the body of
the animal. In the present application, the term "modified fatty
acid" can be regarded as a synthetic or naturally occurring fatty
acid that has been synthetically modified. Also within the context
of this application, the term "organic substituent" refers to
organic chemical structures bonded to the fatty acid that is
effective in decreasing the in vivo rate of .beta.-oxidation of the
fatty acid in tissues of interest.
[0099] The novel FAs described herein are radiolabeled and are
modified with an organic substituent at 2,3; 3,4; 4,5; 5,6 and
other sequence positions. The term "2,3" refers to the carbon bond
between the carbon atoms located at position C2 and C3 (counting
from the carboxyl carbon atom). As such, the related term "3,4"
refers to the carbon atoms at positions C3 and C4. Likewise, the
term "2,3" could also be interpreted by the skilled artisan to
correspond to the term "beta-gamma" in reference to the carbon
atoms, or ".beta..gamma.". The related term "3,4" therefore, could
be interpreted to correspond to the term "gamma-delta" in reference
to the carbon atoms, or ".gamma..delta.". The carboxyl end, of the
molecule will hereinafter be referred to as the "right" end of the
molecule, while the opposite end will be referred to as the "left"
end of the molecule. Compounds can have the general formula, also
depicted in FIGS. 2 and 4:
##STR00006##
[0100] In all of the preceding embodiments, the organic substituent
may be saturated or unsaturated. The organic substituent may also
comprise at least one heteroatom, advantageously N, O or S, most
advantageously O or S. Nitrogen can also be used and it is well
within the skill of the artisan to determine proper substitution of
the nitrogen atom, as well as determining the appropriate
biodistribution of the resultant analog. The carbon backbone of the
fatty acid may also be substituted with at least one heteroatom
herein defined.
[0101] It is further understood that A can be a C.sub.1-C.sub.6
alkyl, alkenyl or akynyl, wherein one or more of the C atoms,
advantageously 1, 2 or 3 C atoms, are substituted by a heteroatom,
advantageously N, O or S, most advantageously O or S. It is well
within the scope of one skilled in the art to determine proper
placement of the nitrogen atom, in addition to determining the
appropriate biodistribution of the resultant analog.
[0102] Further, "aryl" as herein defined may comprise, but is not
limited to, a 5-, 6- or 7-membered ring. Further, "heteroatom" as
herein defined may comprise, but is not limited to, 1, 2 or 3
heteroatoms.
[0103] The chain length of the FA also affects the tissue by which
it is primarily taken up. Generally, a chain length of 12-20 carbon
atoms, inclusive, is optimal for selective uptake by myocardial
tissue, while the liver will selectively take up a chain length of
5-11 carbon atoms, inclusive. The carbon chain of the analog can be
saturated or unsaturated. A preferred embodiment of the invention
is a FA backbone that is saturated and has a cyclic organic
substituent.
[0104] In another embodiment of the present invention, the FA
backbone contains one or more vinyl groups, resulting in
unsaturation of one or more carbon-carbon bonds. Preferably, the
vinyl group or groups are placed on the opposite side of the
carboxyl group, or to the left of the substituent designated by the
letter `A` in the general formula shown above. In another preferred
embodiment, the vinyl groups are appended to the FA backbone such
that the vinylic substituent is branched from the FA chain. More
preferably, 1-6 carbon atoms separate the vinyl group(s) from the
substituent designated by `A`. Generalized drawings of these
preferred embodiments are provided in FIGS. 6 and 8.
[0105] Activation of FAs is an energy-dependent step necessary for
their transport into and sequestration within tissues of interest.
Preferred embodiments of the instant invention are related to
manipulation of the FA metabolism pathway, also referred to herein
as ".beta.-oxidation". This process begins with acyl-coenzyme A
(CoA) synthase, which activates cytosolic free FAs by
decarboxylation of the terminal COOH at the outer mitochondrial
membrane in the presence of adenosirie triphosphate (ATP). This
forms an acyl-adenylate mixed anhydride, which reacts with CoA to
form fatty acyl-CoA and AMP (adenosine monophosphate). One skilled
in the art will know that different isoforms of acyl-CoA synthase
are specific for FAs of varying chain lengths. In the context of
this application, the term "CoA" is used synonymously with
"ScoA".
[0106] Failure to activate FAs to acyl-CoA often results in
"back-diffusion" into the vascular space and as a result, these
back-diffused FAs are not metabolized. However, once activated, FAs
can be "transported" across the inner mitochondrial membranes and
undergo stepwise .beta.-oxidation. Transport across the inner
mitochondrial membrane is also referred to in the context of this
application as "metabolic trapping" or "metabolic retention" of
FAs. Activated fatty acyl-CoA cannot be directly transported across
the inner mitochondrial membranes and the acyl chain must be
transferred to carnitine by an acyl-carnitine/carnitine
transporter. This facilitated diffusion through the inner
mitochondrial membrane is the rate-limiting step for the oxidation
of FAs. During high cardiac workloads or during myocardial
ischemia, esterified FAs are diverted into storage pools as
cytosolic triglycerides and membrane phospholipids. It is
contemplated that activation of the novel analogs described by the
invention and transport into the metabolizing tissue of interest
occurs normally, but subsequent steps relating to disassembly of
the analog are blocked.
[0107] Inside the mitochondrial matrix, FAs are metabolized in four
steps. 1) Formation of a trans-2,3-double bond occurs through
acyl-CoA dehydrogenase from a fatty acyl-CoA precursor to form
trans-.DELTA..sup.2-enoyl-CoA. 2) The trans double bond is then
hydrated by enoyl-CoA hydratase to form 3-L-hydroxyacyl-CoA, which
is subsequently 3) dehydrogenated by 3-L-hydroxylacyl-CoA
dehydrogenase to form .beta.-ketoacyl-CoA. 4) Cleavage of the 2,3
carbon-carbon bond is facilitated through .beta.-ketoacyl-CoA
thiolase to form acetyl-CoA and a new acyl-CoA that is two carbons
shorter than its starting molecule. This process is repeated for
each remaining two-carbon fragment.
[0108] The present invention relates to FAs having a cyclic organic
substituent that cause attenuation of the .beta.-oxidation pathway
by potentially preventing or blocking the metabolic sequence of one
or several ways. For example, the intermediate is not a substrate
for one of the enzymes required during the .beta.-oxidation pathway
or the intermediate cannot undergo a metabolic hydrogenation,
dehydrogenation or hydroxylation step. An early trapping step that
is related to flow and initial uptake may be advantageous for
acquiring blood flow related images. Metabolic trapping or
retention that occurs after one or several metabolic steps can
represent the metabolic integrity of the target tissue. "Reducing"
the formation of metabolic derivatives of the FAs of the invention
can mean total or partial prevention or elimination of metabolic
reactions such as, but not limited to, transport and
back-diffusion, hydrogenation, dehydrogenation, hydroxylation,
ketoacyl formation, and ketoacetyl elimination. As such, one aspect
of the present invention reduces formation of .beta.-ketoacyl-CoA
(step 3). Yet another aspect reduces formation of a
trans-.DELTA..sup.2-enoyl-CoA derivative (step 1). Still another
aspect of the invention reduces formation of the products of
subsequent .beta.-oxidation steps, depending on the organic
substituents and their placement on the FA backbone. The position
of the organic substituent on the FA backbone can determine the
extent of which the molecule undergoes .beta.-oxidation and
metabolism.
[0109] Modifications of the FA, such as the presence of unsaturated
cis-double bonds, can also cause attenuation of FA metabolism. A
cis-3,4-double bond is not a substrate for enoyl-CoA hydratase and
requires enoyl-CoA isomerase, which mediates conversion of the cis
double bond to the more stable, ester-conjugated trans form. Once
this isomerization event occurs, metabolism occurs normally until
the fifth round of .beta.-oxidation, where the presence of another
double bond at an even-numbered carbon atom results in formation of
2,4-dienoyl-CoA. This molecule is a poor substrate for enoyl-CoA
hydratase and requires another enzyme, NADPH-dependent
2,4-dienoyl-CoA reductase. The resultant molecule,
trans-2-enoyl-CoA, is further isomerized to yield trans-3-enoyl-CoA
by 3,2-enoyl-CoA isomerase. Likewise, addition of other organic
substituents, such as branched methyl or phenyl groups, take
advantage of .beta.-oxidation pathway attenuation, which can result
in longer retention times in the metabolizing tissue of
interest.
[0110] An embodiment of the present invention is substitution of an
organic substituent or substituents on the FA chain backbone. This
organic substituent can be placed at 2,3; 3,4; 4,5; 5,6 and other
sequence positions, yielding the addition of the substituent at
positions that are branched from the FA backbone. The position of
the organic substituent on the FA backbone may determine the extent
of which the molecule undergoes .beta.-oxidation and metabolism. In
a preferred embodiment, an organic substituent bonded at the 3,4
position causes the analog to be metabolically trapped in tissues
of interest by substitution of CoA for the carboxyl carbon atom of
the FA analog. The next two steps of metabolism of the resulting
fatty acyl-CoA molecule, formation of trans-.DELTA..sup.2-enoyl-CoA
and 3-L-hydroxyacyl-CoA derivatives, occur normally with
3,4-substitution. However, the resulting 3-L-hydroxyacyl-CoA
derivative is not a substrate for 3-L-hydroxyacyl-CoA dehydrogenase
due to the presence of the organic substituent and thus,
.beta.-ketoacyl CoA formation is prevented. In another preferred
embodiment, an organic substituent bonded at the 2,3 position
causes the analog to be metabolically trapped in tissues of
interest by substitution of CoA for the carboxyl carbon atom of the
FA analog. However, the first metabolic step, which involves
dehydrogenation of the analog to ostensibly form a
trans-.DELTA..sup.2-enoyl-CoA derivative, does not occur, thus
preventing any further metabolism of the analog. It is contemplated
that these specific embodiments can be used advantageously to
measure blood flow.
[0111] Similarly, if an organic substituent is in position 4,5 and
in other subsequent positions on the FA backbone (i.e. 5,6; 6,7;
etc), one or more steps in .beta.-oxidation may be completed and
then be subsequently blocked. Yet another preferred embodiment of
the invention contemplates appending an organic substituent at
positions further on the FA backbone from the carboxyl-terminal
end, such as but not limited to the 4,5; 5,6; 6,7 positions and so
on. It will be apparent to those skilled in the art that the
position of the organic substituent on the FA backbone will
determine the extent of which the molecule undergoes
.beta.-oxidation. In this embodiment, metabolic activity of the
instant invention can be advantageously measured due to its
progression through the .beta.-oxidation pathway.
[0112] Any organic substituent of the analog should be small enough
to permit the formation of the first chemical intermediate involved
in the fatty acid .beta.-oxidation process; too large a substituent
can alter the uptake and behavior of an analog to an undesirable
extent. The chemical nature, as well as the size, of any
substituent can affect the properties of the analog. Generally, an
analog having a substituent which does not render the analog
excessively polar, e.g., an unsubstituted alkyl group, is taken up
primarily by the heart, while an analog containing a polarizing
group, e.g., an alcohol or an ether, will be taken up primarily by
the liver.
[0113] In yet another aspect of the present invention, the
functional group designated by `A` in the general formula provided
above can comprise branched alkyl groups, dimethyl groups, cyclic
alkanes such as cyclopropyl, cyclobutyl, cyclopentyl, or any 3 to
5-membered heterocyclic ring structure. The heterocyclic rings can
contain nitrogen, sulfur or oxygen atoms. A preferred embodiment of
the instant invention is a cyclopropyl ring substituent. Other
preferred embodiments of the invention contemplate cyclobutyl and
cyclopentyl ring substituents, such as those described in the
general diagram of FIG. 10.
[0114] Yet another object of the present invention contemplates
that the organic substituent may be appended onto the FA backbone
in either cis or trans form. A method of cyclopropanation
(Simmons-Smith reaction) is described in the Examples section of
this application and the improved procedure originates from
Charette et al, J. Org. Chem. (1995) 60: 1081. The components of
the reaction can be present as cis or trans isomer, depending on
the FA precursor used for the addition of the cyclopropyl moiety.
The cis diastereomer is preferentially synthesized by using 3
equivalents of the pre-formed complex of Zn(CH.sub.2I).sub.2.DME in
CH.sub.2Cl.sub.2 (see Example 1 for details on this complex) and
incubation for 3 hours at below -10.degree. C., whereas the cis
isomer is made by using the cis olefin and the pre-formed complex
and incubating the mixture for 2 hours at below -10.degree. C. One
skilled in the art will understand that the cyclopropyl group on
the FA backbone has two chiral centers and thus the molecule exists
in two enantiomeric forms: R, R or S, S. An embodiment of the
present invention encompasses each diastereomer and their
respective enantiomers, as well as any racemic mixtures or meso
compounds thereof.
[0115] The novel fatty acids according to the present invention can
be radiolabeled with a positron or gamma-emitting label that is
well known in the art. Preferred embodiments of the invention
comprise .sup.18F, .sup.123I, .sup.131I, .sup.34mCl, .sup.75Br,
.sup.76Br or .sup.77Br. One of ordinary skill in the art will
recognize that different radionuclides display different
pharmacokinetic properties, such as elimination, clearance from
and/or accumulation in biological tissues, and half-life
(t.sub.1/2). Radionuclides are typically synthesized by a
cyclotron, which accelerates subatomic particles from an ion source
along a circular orbit. Particle acceleration inside a chamber is
controlled by two alternating electromagnetic fields. These
accelerated particles can gain energy and collide with a target at
close to the speed of light. Bombardment of particles against the
target result in unstable, radioactive isotopes, which are then
attached to biologically relevant molecules such as those
exemplified by the instant invention. Alternatively, commercially
available radionuclides are widely used and may be appended onto
biologically relevant molecules by chemical synthesis techniques
well known in the art. Typically, the half-lives of radiotracers
used in imaging are relatively short, and thus many cyclotrons are
key features of radiotracer detection apparatuses, such as PET and
SPECT scanners, or gamma cameras. One skilled in the art will
understand the principles of radioisotopic decay and this concept
will not be discussed further in this application.
[0116] A preferred embodiment of the present invention is addition
of a positron or gamma-emitting radiolabel at a position on the FA
backbone that prevents significant loss of the radiolabel during FA
metabolism and migration to other tissues. One of sufficient skill
in the art will appreciate that radiolabeling at the terminal
carboxyl group is not recommended, since this carboxyl group is
removed by specific enzymes during the early metabolic stages of
the FA into the tissues of interest. In one aspect of the
invention, the radiolabel is added to the 9-carbon position of the
FA backbone. In another aspect of the invention, the radiolabel is
added to a terminal phenyl group on the FA backbone located on the
opposing side from the carboxylic acid group. In yet another aspect
of the invention, one or more carbon-carbon bonds on the FA
backbone is unsaturated, resulting in a vinyl group and the
radiolabel is appended directly to the vinyl group. In still
another aspect of the invention, a radiolabeled vinyl group is
appended onto the FA backbone and is branched from the FA
backbone.
[0117] The radiolabeled FAs having a cyclic organic substituent can
be synthesized by traditional organic chemical syntheses well known
in the art. Similarly, the instant invention may be purified and
analyzed by a variety of methods, including column purification,
thin-layer chromatography (TLC), reverse-phase chromatography,
high-performance liquid chromatography (HPLC), gas chromatography
(GC), infrared spectroscopy (IR), nuclear magnetic resonance (NMR)
including variations such as correlation spectroscopy (COSY),
nuclear Overhauser effect spectroscopy (NOESY), and rotating frame
nuclear Overhauser effect spectroscopy (ROESY), and Fourier
Transform, other analytical techniques such as mass spectrometry
(MS) and variations thereof, including electrospray, chemical
ionization, matrix assisted laser desorption ionization,
time-of-flight, fast atom bombardment/liquid secondary ionization,
among many other techniques.
[0118] The pharmaceutical compositions comprising the present
invention, novel radiolabeled FAs that have cyclic organic
substituents, may be in a variety of conventional depot forms.
These include, for example, solid, semi-solid and liquid dosage
forms, such as tablets, pills, powders, liquid solutions or
suspensions, liposomes, capsules, suppositories, injectable and
infusible solutions. Such dosage forms may include pharmaceutically
acceptable carriers and adjuvants, which are well known to those
skilled in the art. These carriers and adjuvants include, for
example, RIBI, ISCOM, ion exchangers, alumina, aluminum stearate,
lecithin, serum proteins, such as human serum albumin, buffer
substances, such as phosphates, glycine, sorbic acid, potassium
sorbate, partial glyceride mixtures of saturated vegetable fatty
acids, water, salts or electrolytes such as protamine sulfate,
disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal
silica, magnesium trisilicate, polyvinyl pyrrolidone,
cellulose-based substances, and polyethylene glycol. Adjuvants for
topical or gel base forms may be selected from the group consisting
of sodium carboxymethylcellulose, polyacrylates,
polyoxyethylene-polyoxypropylene-block polymers, polyethylene
glycol, and wood wax alcohols. The preferred form of the instant
invention is an injectable form. Thus, this form may be subject to
other treatments during preparation to improve their tolerance in
subjects.
[0119] As stated above, the animal of interest, preferably a human
can be injected with the radiolabeled FAs having a cyclic organic
substituent. Any pharmaceutically acceptable dosage route,
including parenteral, intravenous, intramuscular, intralesional or
subcutaneous injection, may be used to administer the novel FA
compositions of the instant invention. For example, the composition
may be administered to the subject in any pharmaceutically
acceptable dosage form including those which may be administered to
a patient intravenously as bolus or by continued infusion over a
period of hours, days, weeks, or months, intramuscularly--including
paravertebrally and periarticularly--subcutaneously,
intracutaneously, intra-articularly, intrasynovially,
intrathecally, intralesionally, periostally, or by oral or topical
routes. Preferably, the compositions of the invention are in the
form of a unit dose and will usually be administered intravenously
by bolus injection.
[0120] The purified FA can be contained in solutions such as
saline, at concentrations suitable for intravenous delivery. These
FAs can be complexed with albumin, a serum protein that binds to
non-esterified FAs in the bloodstream. Typically albumin is added
at a concentration of 2-10%, more preferably between 4-6%.
Alternatively, these FAs can be emulsified in non-ionic detergents
such as but not limited to, polyoxyethylene sorbitan monolaurate
derivatives (Tween), Nonidet P-40, .beta.-D-octylglucoside,
ursodeoxycholic acid(UDCA), or Triton X-100, and resuspended in
solutions containing or lacking albumin prior to injection. The
dosages of radiolabeled cyclic substituted FA must be determined
for each subject prior to administration, and the typical dosage
ranges can be between 0.1-25 mCi, more preferably between 1-5
mCi.
[0121] In yet another aspect, the pharmaceutical compositions
comprising the FAs according to the present invention may also be
administered to any animal, including, but not limited to, horses,
cattle, monkeys, birds, pet animals, such as dogs, cats, birds,
ferrets, hamsters, rodents, squirrels, birds, and rabbits. The
preferred embodiment of the invention is to monitor diseases or
disease states associated with blood flow, FA metabolism or for
imaging an organ of interest in a human.
[0122] The present invention further comprises methods to measure
and/or identify changes in blood flow and metabolism in tissues of
interest in response to disease states, exercise, pharmacological
agents, or for diagnostic imaging.
[0123] In one embodiment of the instant invention, a method of
measuring blood flow in a subject comprises the following steps:
[0124] a) localizing a detectable amount of the FA composition of
the invention, to a tissue of interest; [0125] b) detecting a
signal from said radiolabeled FA composition in a tissue of
interest within about 1 minute and about 5 minutes after
administration; [0126] c) imaging a tissue of interest and; [0127]
d) determining the rate of blood flow in a tissue of interest.
[0128] The present invention further provides a method of measuring
metabolism in a subject comprising the following steps: [0129] a)
localizing a detectable amount of the FA composition of the
invention, to a tissue of interest and; [0130] b) detecting a
signal from said radiolabeled FA composition in a tissue of
interest within about 30 minutes and about 120 minutes after
administration; [0131] c) imaging a tissue of interest and; [0132]
d) determining the rate of metabolism in a tissue of interest.
[0133] Another embodiment of the present invention provides a
method for retaining a fatty acid composition of the invention in a
tissue of interest, comprising the steps of: [0134] a) localizing a
detectable amount of the composition to the tissue; [0135] b)
retaining the composition, or a metabolic derivative thereof, in
the tissue by reducing transport and back-diffusion of the
composition; and [0136] c) detecting the composition or the
metabolic derivative in the tissue.
[0137] In another embodiment of the present invention, a method for
retaining a fatty acid composition of the invention in a tissue of
interest, comprising the steps of: [0138] a) localizing a
detectable amount of the composition to the tissue; [0139] b)
retaining the composition, or a metabolic derivative thereof, in
the tissue by reducing dehydrogenation of the composition; and
[0140] c) detecting the composition or the metabolic derivative in
the tissue.
[0141] The present invention further provides a method for
retaining a fatty acid composition of the invention in a tissue of
interest, comprising the steps of: [0142] a) localizing a
detectable amount of the composition to the tissue; [0143] b)
retaining the composition, or a metabolic derivative thereof, in
the tissue by reducing hydroxylation of the composition; and [0144]
c) detecting the composition or the metabolic derivative in the
tissue.
[0145] Another embodiment of the present invention provides a
method for retaining a fatty acid composition of the invention in a
tissue of interest, comprising the steps of: [0146] a) localizing a
detectable amount of the composition to the tissue; [0147] b)
retaining the composition, or a metabolic derivative thereof, in
the tissue by reducing ketoacyl formation of the composition; and
[0148] c) detecting the composition or the metabolic derivative in
the tissue.
[0149] Yet another embodiment of the present invention provides a
method for retaining a fatty acid composition of the invention in a
tissue of interest, comprising the steps of: [0150] a) localizing a
detectable amount of the composition to the tissue; [0151] b)
retaining the composition, or a metabolic derivative thereof, in
the tissue by reducing ketoacetyl elimination of the composition;
and [0152] c) detecting the composition or the metabolic derivative
in the tissue.
[0153] Numerous methods by which one may use the present invention
in imaging, measurements of blood flow, and FA metabolism are
contemplated, used singularly, or in combination with other imaging
modalities. For example, cardiac imaging modalities generally
measure two parameters: blood flow (perfusion) and myocardial
viability (metabolism). These measurements are useful in numerous
cardiac diseases. Animal models of measuring blood flow often use
microspheres (Miller, D. D., et al. (1988) Circ. Res. 63: 681-692),
however their use is precluded in humans, as microspheres can block
circulation. Traditional methods of observing myocardial perfusion
and metabolism have utilized two different tracers each separately
used to measure either blood flow or metabolism. Widely used
markers for blood flow are exemplified by .sup.201Thallium
(.sup.201Tl), .sup.99mTechnetium (.sup.99mTc), .sup.13N-Ammonia,
and .sup.82Rubidium. However, unlike .sup.201Tl, .sup.99mTc,
.sup.13N-Ammonia, and .sup.82Rubidium, the uses of which are only
limited to measurements of perfusion, certain FA tracers are able
to measure both flow and metabolism (Poe, N. D. et al. (1977)
Radiology 124: 419-424; van der Wall, E. E. et al. (1980) Eur. J.
Nucl. Med. 5: 401-405; Kairento, A. L. et al. (1988) Nucl. Med.
Biol. 15: 333-338; Kawamoto, M. et al. (1994) J. Nucl. Cardiol. 1:
522-8; Kobayashi, H. et al. (1997) J. Nucl. Med. 39: 1117-1122;
Miller, D. D., et al. (1988) Circ. Res. 63: 681-692).
[0154] Similarly, the instant invention can be used in a variety of
methods to answer a specific diagnostic question. By way of
example, methods can include administration of the invention under
conditions of rest and/or stress induced by exercise, disease
states, or pharmacological agents. Likewise, the radiolabeled FAs
having a cyclic organic substituent can also be used in sequential
imaging experiments, depending on the radioisotope used. Blood flow
to an organ of an animal can be determined within 1 second to about
10 minutes, preferably between about 1 minute and about 5 minutes
after the radiolabeled cyclic substituted FA is administered to the
animal. Metabolism by the organ of interest can be determined
within a time period between about 10 minutes to about 24 hours,
preferably between about 30 minutes and about 120 minutes after
administration of the radiolabeled FA into the bloodstream of the
animal.
[0155] Nuclear cardiac imaging using the present invention can be
used to detect a wide variety of cardiac derangements. One of
sufficient skill in the art will recognize that cyclic substituted,
radiolabeled FAs can be used singularly as a marker for blood flow
and for cardiac metabolism, or may be used in combination with
another tracer. Such tracers include, but are not limited to
.sup.13N-Ammonia, .sup.57Co-Cyanocobalamin, .sup.59Fe-Ferrous
Citrate, .sup.18F-Fluorodeoxyglucose, .sup.67Ga-Gallium Citrate,
.sup.111In-Indium Oxyquinoline, .sup.111In-Indium Pentetate,
.sup.111In-Indium Pentetreotide, .sup.111In-Indium Satumornab
Pendetide, Radioiodinated Iobenguane, .sup.123I-Iodohippurate
Sodium, .sup.131In-Iodohippurate Sodium, .sup.123I-Iofetamine,
.sup.125I-Iothalamate Sodium, .sup.81Krypton, .sup.11C-Methionine,
RadioiodinatedAlbumin, .sup.82Rubidium, Sodium .sup.51Chromate,
Sodium .sup.18-Fluoride, Sodium .sup.123Iodide, Sodium
.sup.131Iodide, Sodium .sup.99m-Pertechnetate, .sup.99mTc-Albumin,
.sup.99mTc-Albumin (Aggregated), .sup.99mTc-Albumin (Colloidal),
.sup.99mTc Arcitumomab, .sup.99mTc-Bicisate, .sup.99mTc-Disofenin,
.sup.99mTc-Exametazime, .sup.99mTc-Gluceptate,
.sup.99mTc-Lidofenin, .sup.99mTc-Mebrofenin, .sup.99mTc-Medronate,
.sup.99mTc Mertiatide, .sup.99mTc-Nofetumomab Merpentan,
.sup.99mTc-Oxidronate, .sup.99mTc-Pentetate,
.sup.99mTc-Pyrophosphate, .sup.99mTc-(Pyro- and trimeta-)
Phosphates, .sup.99mTc Sestamibi, .sup.99mTc Succimer,
.sup.99mTc-Sulfur (Colloidal), .sup.99mTc-Teboroxime,
.sup.99mTc-Tetrofosmin, .sup.201Thallous Chloride, .sup.127Xenon,
.sup.133Xenon. It will be apparent that not all of the
aforementioned tracers are suitable for cardiac imaging and that
use of the instant invention is not necessarily limited to those
related to cardiovascular disease.
[0156] The tissues of interest can be any tissues that utilize FAs
as a source of energy. The tissues can be, but are not limited to,
cardiac tissue, brain, liver, bone, spleen, lung, blood, kidney,
gastrointestinal, muscle, and adrenal tissue. A preferred
embodiment of the present invention is cardiac tissue. Another
preferred embodiment can also encompass liver tissue.
[0157] Cardiac diseases or disease states that can be monitored
using radiolabeled FAs having cyclic organic substituents include,
but are not limited to, acute myocardial infarction, unstable
angina, chronic ischemic heart disease, coronary artery disease,
myocarditis, dilated, hypertrophic, and restrictive
cardiomyopathies, congenital heart diseases, hypertensive heart
disease, post-transplant heart disease, allograft vasculopathies,
valvular heart disease, and pharmacologically induced conditions
such as doxorubicin cardiotoxicity. It is contemplated that methods
of use of radiolabeled FAs exemplified by the present invention
will be modified according to the particular disease examined.
[0158] The instant invention can also be used in numerous
non-cardiac disease states described herein, such as: abscess and
infection; biliary tract blockage; blood volume studies; blood
vessel diseases; blood vessel diseases of the brain; bone diseases;
bone marrow diseases; brain diseases and tumors; cancer and
neoplasms; colorectal disease; diabetes; disorders of iron
metabolism and absorption; heart disease; heart muscle damage such
as infarction and isehemia; impaired flow of cerebrospinal fluid in
brain; kidney diseases; liver diseases; lung diseases; parathyroid
diseases and/or parathyroid cancer; pernicious anemia and/or
improper absorption of vitamin B.sub.12 from intestines; red blood
cell diseases; salivary gland diseases; spleen diseases; stomach
disorders and intestinal bleeding; tear duct blockage, thyroid
diseases and/or thyroid cancer, urinary bladder diseases. A
preferred embodiment of the present invention is its use in
detecting cardiac myopathies by measuring blood flow and FA
metabolism. It is understood that diagnosis of the aforementioned
diseases will often require the use of other radiotracers, also
described above. It is apparent that use of the instant invention
is not only limited to detection of diseased states, but also for
diagnostic imaging in healthy subjects.
[0159] One of ordinary skill in the art will appreciate the
numerous methods of detecting cardiac imaging agents exemplified by
the present invention, as well as understand that different
radionuclides will require different detection techniques. Positron
Emission Tomography (PET) and Single Photon Emission Computed
Tomography (SPECT) are imaging techniques in which a radionuclide
is synthetically introduced into a molecule of potential biological
significance, such as a radiolabeled FA indicated by the instant
invention. The subsequent uptake of the radiotracer is measured
over time and used to obtain information about the physiological
process of interest. While PET and SPECT rely on similar principles
to produce their images, important differences in instrumentation,
radiochemistry, and experimental applications are dictated by
inherent differences in their respective physics of photon
emission.
[0160] Unstable nuclides that possess an excess number of protons
may take one of two approaches in an effort to reduce their net
nuclear positivity. In one radioactive decay scheme, a proton is
converted to a neutron and a particle called a positron is emitted
(Hoffman, E. J., and Phelps, M. E. New York: Raven Press; 1986:
237-286; Sorenson, J. A., and Phelps, M. E. Philadelphia: W.B.
Saunders; 1987). Of identical mass but opposite charge, positrons
are the antimatter equivalent of electrons. When ejected from the
nucleus, a positron collides with an electron, resulting in the
annihilation of both particles and the release of energy. Two y
photons are produced, each of equivalent energy and opposite
trajectory (generally 180.degree. apart).
[0161] The unique spatial signature of back-to-back photon paths is
exploited by PET scanners in locating the source of an annihilation
event, a method known as coincidence detection (Hoffman, E. J., and
Phelps, M. E. New York: Raven Press; 1986: 237-286; Links, J. M.
New York: Raven Press; 1990: 37-50). PET (and SPECT) scanners
employ scintillation detectors made of dense crystalline materials
(e.g., bismuth germanium oxide, sodium iodide, or cesium fluoride),
that capture the high-energy photons and convert them to visible
light. This brief flash of light is converted into an electrical
pulse by an adjacent photomultiplier tube (PMT). The crystal and
PMT together make up a radiation detector. A PET camera is
constructed such that opposing detectors are electronically
connected. Thus, when separate scintillation events in paired
detectors coincide, an annihilation event is presumed to have
occurred at some point along an imaginary line between the two.
This information is used to reconstruct images using the principles
of computed tomography. Conversely, single events are ignored.
Although it is conceivable that two unrelated photons from
spatially separate annihilation events might reach opposing
detectors in unison, these accidental coincidences are much less
frequent than true ones. Nevertheless, random coincidences
constitute a source of background noise in PET images (Hoffman E J
et al. J Comput Assist Tomogr (1981); 5:391-400; Hoffman, E. J.,
and Phelps, M. E. New York: Raven Press; (1986): 237-286; Links, J.
M. New York: Raven Press; (1990): 37-50).
[0162] The skilled artisan will recognize the intrinsic limitations
of PET derive from the nature of positron decay and the principle
of coincidence detection. Specifically, PET recognizes the site of
positron annihilation, which does not necessarily coincide with the
site of radioactive decay. Annihilation often occurs some distance
away from the positron's origin. The distance separating these two
events, decay and annihilation, depends on the average kinetic
energy of the positron as it leaves the nucleus, and varies
according to the specific isotope involved (Phelps M E, et al. J
Nucl Med (1975); 16: 649-652). In addition, if the positron is not
entirely at rest at annihilation, photons will be emitted at an
angle slightly different than 180.degree.. Taken together, remote
positron annihilation and photon non-colinearity place a
theoretical limit on PET's achievable spatial resolution (Links J
M. New York: Raven Press; (1990): 37-50).
[0163] In another embodiment of the present invention, isotopes
that decay by electron capture and/or .gamma. emissions can be
directly detected by SPECT. Certain proton-rich radionuclides, such
as .sup.123I and .sup.99mTc, may instead capture an orbiting
electron, once again transforming a proton to a neutron (Sorenson J
A, and Phelps M E. Philadelphia: W.B. Saunders; 1987). The
resulting daughter nucleus often remains residually excited. This
meta-stable arrangement subsequently dissipates, thereby achieving
a ground state and producing a single y photon in the process.
Because y photons are emitted directly from the site of decay, no
comparable theoretical limit on spatial resolution exists for
SPECT. However, instead of coincidence detection, SPECT utilizes a
technique known as collimation (Jaszczak R J. Boca Raton: CRC
Press; (1991): 93-118). A collimator may be thought of as a lead
block containing many tiny holes that is interposed between the
subject and the radiation detector. Given knowledge of the
orientation of a collimator's holes, the original path of a
detected photon is linearly extrapolated and the image is
reconstructed by computer-assisted tomography.
[0164] It is contemplated that use of the instant invention will
require processing and analysis of data obtained from imaging
modalities such as but not limited to PET or SPECT. The following
mathematical algorithms can determine the rate of uptake of novel
radiolabeled FA analogs having a cyclic organic substituent. Other
mathematical models can also be similarly employed and/or modified
to determine the rate of blood flow and metabolic activity
(Sokoloff, L. et al (1977) J. Neurochem. 28: 879-916; Elmaleh, D.
R. et al. (1994) J. Nucl. Med. 35: 496-503; Marshall, R. C. et al.
(1998) Am. J. Physiol. 275: H668-H679; Wieler, H. et al. (1990)
Nuc. Med. Commun. 11: 865-878). The collected early and late images
in tissues of interest can be manipulated to obtain values that may
indicate abnormalities.
[0165] Mathematical modeling can be used to assess the rate of
uptake of FA analogs of the present invention. There is a
reproducible relationship between the net extraction fractions of
native FA and FA analog. Assuming the uptake and metabolism of
native FA is in a steady state, the rate of metabolism, defined as
R for purposes of this application, can be deduced from the Fick
equation:
R=E.sub.n*F*Ca (mol/min/g) [0166] where [0167] E.sub.n=net
extraction fraction [0168] F=blood flow (ml/min/g), which can be
estimated from the early images. [0169] Ca=arterial concentration
of FA (mol/ml).
[0170] The assumption is such that at steady state, the rate at
which FA leaves the tissue through oxidation must be equal to the
rate at which FA is entering the tissue from the blood. The net
extraction of a substance metabolized by the heart in a steady
state can be determined from the arterial or arterialized blood and
venous blood concentrations of FAs.
[0171] A relationship between the net extraction of FA analog and
FA, E.sub.n(FA analog), can be used to calculate the rate of FA
metabolism, R. We can write this relationship as
L=E.sub.n/E.sub.n(FA analog)
[0172] L here plays the same role as the "lumped constant" in
Sokoloff's model for deoxyglucose metabolism (Sokoloff, L. et al
(1977) J. Neurochem. 28: 879-916). The difference between the two
models is that L may not be constant. `L` can vary with blood flow,
free fatty acid concentration, FFA composition, or other
physiological parameters. The behavior of L is best understood by
performing a series of experiments to determine the relationship
between E.sub.n and F.sub.n[FA analog] in different physiological
states.
[0173] Basic modeling assumptions are such that 1) fatty acid
uptake and metabolism is in a steady state. In other words, all the
FA pools and reaction products have constant concentrations. This
assumes that the net rate at which FA is removed from the blood
equals the rate of FA catabolism. (2) Labeled FA analog is
generally present in trace amounts, so that its presence does not
alter the FA steady state. The various transport and chemical
reaction rates for FA analog can then be expected to depend
linearly on the concentrations of the FA analogs. This modeling
approach accounts for capillary-tissue exchange as well as for
intracellular metabolism of the administered FA analog.
Intracellular metabolism of native fatty acids is assumed to create
gradients, which regulate the access of FA analog to the
intracellular space. Coupled to these metabolically induced
gradients are the effects of blood flow and capillary transport
properties. In this approach, flow and transport mechanisms are
included in capillary tissue exchange while the metabolic processes
are included in intracellular metabolic parameters.
[0174] In general, the transport of any substance between capillary
and tissue depends on two factors: (i) Intrinsic capillary
transport rate for a freely diffusing substance (or the
permeability surface area product; for carrier mediated transport,
it is a function of "apparent" Michaelis-Menten parameters that
describe the transport process). In a "microscopic" modeling
approach, a protein-substrate complex would characterize each
barrier, but here they are lumped together into a single "apparent"
rate parameter (k). (ii) Local blood flow per ml of tissue. Blood
flow and transport rate can be thought to describe two competing
processes, one carrying molecules through the capillary wall, the
other carrying them out the venous end of the capillary. These two
rates are combined to give extraction fraction E, the fraction of
molecules entering a capillary bed, which pass through the wall of
the capillary at least once. As used here, E is the one-way
(unidirectional) extraction fraction, not the net extraction
fraction.
[0175] It is assumed that an FA analog cannot undergo
.beta.-oxidation, and hence, is either trapped in mitochondria or
incorporated in the triglyceride pool. The triglyceride pool turns
over very slowly. With respect to FA analog, the concentration of
FA analog in myocardial tissue, following IV injection, can reach a
plateau after ten to fifteen minutes. The simplest model consistent
with the measured tissue curves and the biochemical data is one in
which tissue is described by two compartments, a precursor FFA pool
and a metabolically trapped pool, both driven in response to the FA
analog concentration in the blood plasma.
[0176] Because FA analogs cannot undergo .beta.-oxidation and
because the turnover rate for the triclyceride pool is slow, the
plateau of the tissue curve can reflect both components.
Measurements with FA analogs can only determine the rate at which
FA analog passes through the committed step to acyl-CoA.
Accordingly, the steady state rate for incorporation into
triglyceride plus .beta.-oxidation can be measured. This situation
is analogous to the result that would be obtained if Fick-type
atrial/ventricular measurements with a native fatty acid such as
palmitate could be made.
[0177] Based on the considerations outlined above, an operational
equation capable of describing the PET measurements in terms of the
blood flow is described, F being the unidirectional extraction
fraction, E as the net extraction fraction, E.sub.n, and the rate
K, with which FA analog is cleared from the "precursor pool". The
rate constant K is the sum of two rates, K.sub.2 the rate of
back-diffusion, and K.sub.3, the rate of activation of FA analog to
the CoA form. Neglecting tracer in blood, the operational equation
is given as
C(t)=F*[(E=E.sub.n)*Exp(-K*t)+E.sub.n](*)Ca(t) [0178] Where C(t) is
the tissue concentration at time t, [0179] Ca(t) is the plasma
concentration at time t and [0180] (*) represents the mathematical
operation of convolution.
[0181] For the model of this invention, E.sub.n=E*K3/K2+K3. This
expression has a simple interpretation-i.e., E is the probability a
FA analog molecule will leave the blood on a single capillary
transit, K3/(K2+K3) is the probability that a FA analog molecule
entering the tissue will be metabolically trapped. No assumptions
were made in the derivation of the operational equation limiting
the value of E, other than it be in the range (0.1). It should also
be noted that the operational equation is formally similar to that
of Sokoloff.
[0182] Under the assumption that a steady state was established
with respect to the consumption of FA analog, where the plasma FA
content was the concentration of FA analog, then the analog's
metabolic rate would be F*E.sub.n[FA analog]. Therefore, the
quantity F*E.sub.n can be interpreted as the analog utilization
rate to a unit quantity of FA analog in steady state.
[0183] Analysis of the operational equation of the method shows
that as t (or, the plateau concentration is given by
F*E.sub.n*.intg. of the plasma concentration curve). Thus, the
tissue concentration at the plateau is directly proportional to the
metabolism of the analog.
[0184] A better understanding of the instant invention and of its
many advantages will be facilitated via the following examples,
which further describe the invention and given by way of
illustration. The examples that follow are not to be construed as
limiting the scope of the invention in any manner. In light of the
present disclosure, numerous embodiments within the scope of the
claims will be apparent to those ordinary skill in the art.
EXAMPLES
Example 1
Synthesis of .sup.18F-labeled 3,4-cyclopropyl-heptadecanoic acid
([.sup.18F]-FCPHA)
[0185] Synthesis of [.sup.18F]-FCPHA was performed using the
following method. PCC (pyridinium chlorochromate; 24.8 g, 115.2
mmol) was slowly added in portions to a solution of
6-benzyloxy-1-hexanol (20 g, 96 mmol) in 200 ml of methylene
chloride at room temperature (25.degree. C., RT). The black mixture
was stirred for 2 hours, and filtered through 50 g of silica gel.
The solvent was removed and the crude oil was separated
chromatographically on silica gel using a mixture of hexane:ethyl
acetate at a ratio of 90:10. This yielded the aldehyde equivalent,
6-benzyloxy-1-hexanal, at a scale of 14 g (70%).
[0186] Under a nitrogen atmosphere, octyl bromide (12 g, 115 mmol)
in anhydrous ether (60 mL) was added to magnesium metal (2.8 g, 116
mmol) in 10 ml of ether at a rate as to maintain gentle reflux.
After addition, the reaction mixture was stirred for 1 hour and
then 6-benzyloxyhexanal (12 g, 58 mmol) in 20 ml of ether was added
dropwise to the reaction mixture at RT. The mixture was stirred for
4 hours and poured over ice water. Acidification was achieved with
10% hydrochloric acid (HCl), then extracted with ether. The
combined extracts were washed with brine, dried, and the solvent
removed. Chromatography of the crude oil on silica gel using
hexane:ethyl acetate at a ratio of 80:20 yielded 16.5 g, or 89%, of
the alcohol. The chemical shift of the compound, detected by
.sup.1H-NMR, was as follows: .sup.1H NMR (CDCl.sub.3) .delta. 0.89
(t, 3H, CH.sub.3), 1.2-1.7 (m, 22H, CH.sub.2), 3.45 (m, 2H,
CH.sub.20), 3.55 (m, 1H, CH--O), 4.55 (s, 2H, CH.sub.2--O), 7.35
(m, 5H, benzyl).
[0187] The resulting compound, 1-benzyloxy-6-tetradecanol (16 g),
was hydrogenated overnight using 100 mg of 10% palladium on
activated carbon (Degussa A G; Frankfurt, Germany) in 100 ml of
methanol to yield 1,6-tetradecandiol (11.1 g, 4.85 mmol). This diol
compound, in 100 ml of methylene:pyridine (9:1), was treated with
trityl chloride (13.5 g, 4.85 mmol). The mixture was stirred for 4
hours, then washed with ice-cold 10% HCl until the aqueous phase
was acidic. Washing in brine then followed this until the aqueous
phase was neutral. Solvent removal and chromatography on silica gel
in hexane:ethylacetate (80:20) of the oil gave 20.1 g (87%) of the
trityl alcohol. The chemical shift of the compound, detected by
.sup.1H-NMR, was as follows: .sup.1H-NMR (CDCl.sub.3) .delta. 0.89
(t, 3H, CH.sub.3), 1.2-1.7 (m, 22H, CH.sub.2), 3.05 (m, 2H,
CH.sub.2O), 3.55 (m, 1H, CH--O), 3.55 (m, 1H, CH--O), 7.2-7.5 (m,
15H, benzyl).
[0188] The resulting compound, 1-trityl-6-tetradecanol (20 g, 42.3
mmol), was slowly mixed with 60% sodium hydride (2 g, 50 mmol) and
benzyl bromide (7.2 g, 42.3 mmol) in dry dimethylformamide (DMF;
100 ml) in a water bath set at RT. The mixture was stirred for 4
hours at RT and poured into ice water, then extracted with ether.
Chromatography of the crude oil on silica gel using hexane:ethyl
acetate (90:10) yielded 19 g (34 mmol), or 80% of the diether
compound, 6-benzyloxy-1-trityl-tetradecane diether. This diether
compound was mixed with 100 mg of p-toluene sulfonic acid in
methanol (100 L) at RT for 4 hours. Sodium bicarbonate (100 mg) was
added and the solvent removed by rotary evaporation. The oil was
separated by chromatography on silica gel in hexane:ethyl acetate
(80:20) yielded 7.5 g, or 90% of the alcohol. The chemical shift of
the resulting compound, 6-benzyloxy-1-tetradecanol, is as follows:
.sup.1H-NMR (CDCl.sub.3) .delta. 0.89 (t, 3H, CH.sub.3), 1.2-1.7
(m, 22H, CH.sub.2), 3.38 (m, 2H, CH.sub.2O), 3.55 (m, 1H, CH--O),
4.7 (s, 2H, CH.sub.2--O), 7.35 (m, 5H, benzyl).
[0189] PCC (9.7 g, 45 mmol) was added in portions to a solution
containing 6-benzyloxy-1-tetradecanol (7.5 g, 30 mmol) in methylene
chloride (100 ml) at RT. The black mixture was stirred for 2 hours,
then filtered through a bed of silica gel using hexane:ethyl
acetate (90:10). The corresponding aldehyde,
6-benzyloxy-tetradecanal, was produced at a yield of 6.1 g (81%).
This aldehyde compound was mixed with carbethoxymethylene
triphenylphosphorane (10 g, 29 mmol) in 100 ml of methylene
chloride at RT. The mixture was allowed to stir overnight, after
which the solvent was removed and replaced with hexane. Solid
precipitates were removed by filtration. Following chromatography
on silica gel using hexane:ethyl acetate at a ratio of 90:10, the
corresponding ester was detected at 8.4 g scale (90%). The chemical
shift of the ester, 8-benzyloxyhexadec-2-enoate ethyl ester, is as
follows: .sup.1H-NMR (CDCl.sub.3) .delta. 0.89 (m, 6H, CH.sub.3),
1.2-1.5 (m, 20H, CH.sub.2), 2.2 (m, 2H, CH.sub.2--C.dbd.), 3.35 (m,
2H, CH.sub.2O), 4.2 (q, 2H, MeCH.sub.2--O), 4.55 (dd, 2H,
CH.sub.2--O), 5.8 (d, 1H, CH.dbd.C), 6.95 (d,t 1H, CH.dbd.C), 7.35
(m, 5H, benzyl).
[0190] DIBAL (diisobutylaluminum hydride; 8 ml, 44 mmol) in 40 ml
hexane, was added dropwise to the ester compound
8-benzyloxyhexadec-2-enoate ethyl ester (8.4 g, 22 mmol) in 40 ml
methylene chloride at -78.degree. C. The mixture was allowed to
reach 0.degree. C. and 5 ml of ethyl acetate was added dropwise,
followed by ice. The resultant slurry was acidified with 10% HCl,
followed by ether extraction. The combined ether extracts were
washed with brine, dried, and the solvent was removed. Separation
of the crude oil by silica gel chromatography using hexane:ethyl
acetate (80:20) yielded 6.7 g, or 90% of the unsaturated primary
alcohol.
[0191] Addition of the cyclopropyl moiety was performed essentially
as described by Charette and coworkers (Charette et al, J. Org.
Chem. 1995 60: 1681). A 100-ml round bottom flask containing 40 ml
of dry methylene chloride was cooled to -25.degree. C. and placed
under a nitrogen atmosphere. To this flask, 3.8 ml of diethyl zinc,
followed by 3.9 ml of 1,2-dimethoxyethane (DME), was added.
Diiodomethane (6 ml) was added dropwise to the solution, while
maintaining the reaction temperature between -25.degree. C. and
-10.degree. C. This solution was then added by double-ended needle,
to a solution containing 8-benzyloxyhexadec-2-enol (2 g, 5.8 mmol),
dioxaborolane (made from (+)N,N,N',N'-tetramethyl-L-tartaramide and
butylboronic acid), and 300 mg of 4 .ANG. molecular sieves in
methylene chloride (40 ml) under nitrogen at -40.degree. C. and
-30.degree. C. The reaction mixture was stirred for 2 hours at
-25.degree. C., then allowed to warm to 0.degree. C. Saturated
ammonium chloride in an 80 ml volume was added to the mixture, then
stirred at RT for 16 hours. Solid matter was removed by filtration
and the layers were separated. The aqueous layer was extracted with
methylene chloride, while organic layers were pooled, washed with
water, and dried. The crude oil was separated by silica gel
chromatography in hexane:ethyl:acetate (90:10), yielding 1.9 g
(90%) of the cyclopropyl alcohol. This reaction was repeated twice.
The chemical shift of the molecule,
8-benzyloxy-2,3-cyclopropylhexadecanol, is as follows: .sup.1H-NMR
(CDCl.sub.3) .delta. 0.3 (m, 2H, ring CH.sub.2), 0.5 (m, 1H, CH),
0.75 (m, 1H, CH), 0.89 (t, 3H, CH.sub.3), 1.2-1.6 (m, 22H,
CH.sub.2), 3.35 (m, 1H, CHO), 3.45 (m, 2H, CH.sub.2--O), 4.55 (s,
2H, CH.sub.2-0), 7.35 (m, 5H, benzyl). Addition of dioxaborolane in
this reaction will form the S,S isomer as the predominant species.
The reaction can be performed without dioxaborolane to yield the
racemic (R,R; S,S) mixture.
[0192] Iodine (6 g, 24 mmol) was added in portions to a solution
containing 8-benzyloxy-2,3-cyclopropylhexadecanol (5.7 g, 15.8
mmol) and triphenylphosphine (6.2 g, 23.7 mmol) in 100 ml of
dimethylformamide. The dark red mixture was combined with 100 ml of
10% sodium thiosulfate and extracted with ether. The combined
extract was washed with water, brine, and the solvent was
subsequently removed. Silica gel chromatography in hexane:ethyl
acetate (95:5) yielded 4.5 g (60%) of the alkyl iodide. This alkyl
iodide, 8-benzyloxy-1-iodo-2,3-cyclopropylhexadecane (4.5 g, 9.6
mmol), and sodium cyanide (0.95 g, 19.2 mmol) in 60 ml of dimethyl
sulfoxide (DMSO), was heated to 80.degree. C. for 2 hours. The
mixture was poured into 100 ml of water, followed by ether
extraction. The combined extract was washed with water, dried, and
solvent removed. The crude oil was separated by silica gel
chromatography in hexane:ethyl, acetate (80:20) and yielded 3.0 g
(87%) of the corresponding nitrite compound. Chemical analysis by
.sup.1H-NMR resulted in: .sup.1H NMR (CDCl.sub.3) .delta. 0.3 (m,
2H, ring CH.sub.2), 2.4 (m, 2H, CH.sub.2--CN), 3.35 (m, 1H, CHO),
4.55 (s, 2H, CH.sub.2--O), 7.35 (m, 514, benzyl).
[0193] A mixture containing the above-described nitrite compound,
9-benzyloxy-2,3-cyclopropylhexadecane nitrite (3.0 g, 8.1 mmol),
600 mg of potassium hydroxide (KOH), and 3 drops of water in 40 ml
ethylene glycol, was heated for 6 hours at 170.degree. C. Once
cooled, the mixture was diluted with 80 ml of 10% HCl, then
extracted with ether. The combined extract was dried and the
solvent was removed. The crude carboxylic acid was treated with
diazomethane (generated from N-methyl-N'-nitro-N-nitrosoguanidine
and 40% KOH in ether). The reaction mixture was stirred for 1 hour
before solvent removal. Chromatography of the crude oil on silica
gel with hexane:ethyl acetate (80:20) gave 1.9 g, or 60%, of the
corresponding ester, 9-benzyloxy-3,4-cyclopropylheptadecanoate
methyl ester (4.7 mmol). This ester was hydrogenated overnight with
25 mg of 10% palladium on activated charcoal in 50 ml of methanol.
Hydrogenation yielded 1.3 g, or 89% of the hydroxy ester. Chemical
analysis resulted in the following: .sup.1H-NMR (CDCl.sub.3)
.delta. 0.30 (d,d, 2H, ring CH.sub.2), 0.53 (m, 1H, CH), 0.75 (m,
1H, CH), 0.89 (t, 3H, CH.sub.3), 1.2-1.6 (m, 22H, CH.sub.2), 2.23
(m, 2H, CH.sub.2--CO), 3.58 (m, 1H, CHO), 3.69 (s, 3H,
CH.sub.3).
[0194] The hydroxy ester, 9-hydroxy-3,4-cyclopropylheptadecanoate
methyl ester (0.5 g, 1.6 mmol), and methane sulfonyl chloride (0.2
g, 1.76 mmol), and 10 mg of DMAP (4-dimethylaminopyridine) in 20 ml
of methylene chloride:pyridine (90:10), was stirred for 2 hours.
The mixture was washed with 10% HCl until the aqueous layer was
acidified, then washed again with 10% NaHCO.sub.3. Chromatographic
analysis of the crude oil on silica gel, in methylene
chloride:methanol (95:5) gave 0.5 g (83%) of the mesylate ester,
9-hydroxy-3,4-cyclopropylheptadecanoate methyl ester.
[0195] The radiolabel .sup.18F, contained in water (60 mCi, 1 ml),
was added to a vial containing 10 mg of Kryptofix-222
(4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane; Merck,
Whitehouse Station, N.J.) and 4 mg of K.sub.2CO.sub.3. Water was
removed using a stream of nitrogen at 115.degree. C., followed by
the three rounds of addition of acetonitrile (2 ml). The mesylate
ester, 9-hydroxy-3,4-cyclopropylheptadecanoate methyl ester (10 mg)
in 1 ml of acetonitrile was combined with the radiolabel mixture
and heated for 10 minutes in 120.degree. C. This yielded the
.sup.18F-labeled ester at 80% yield after silica Sep-Pak
purification (Waters Corporation, Milford, Mass.) in hexane:ethyl
acetate (85:15). The labeled ester was placed in a vial and the
solvent was removed. It was replaced with 0.1 ml of 1M lithium
hydroxide (LiOH) and 0.3 ml of methanol. The reaction vial was
heated to 60.degree. C. for 20 minutes. Methanol was removed by
nitrogen stream at 60.degree. C. The solution was acidified in 10
ml of 10% HCl, and the mixture was extracted twice with 10 ml of
ether. The combined ether extract was passed through a silica
Sep-Pak chromatography cartridge and solvent was removed, affording
13 mCi of the final product,
9-[.sup.18F]-fluoro-3,4-cyclopropylheptadecanoic acid. Radio
thin-layer chromatography (TLC) showed 94% radiochemical purity
(silica gel plate, 25:75 ethyl acetate/hexane, R.sub.f=0.40) with
the remaining activity as unhydrolyzed ester (silica gel plate,
15:85 ethyl acetate/hexane, R.sub.f=0.80). A schematic
representation of the organic synthesis described above is shown in
FIG. 1. The labeled fatty acids were formulated in 10% ethanol in
saline for rat studies and 4% BSA in saline for monkey studies
(sterile filtered through a 0.22 .mu.m Millipore filter).
Example 2
Biodistribution of .sup.18F-labeled 3,4-cyclopropyl-heptadecanoic
acid([.sup.18F]-FCPHA)
[0196] The initial myocardial behavior of [.sup.18F]-FCPHA was
essentially similar to that of normal FAs, as evidenced that this
analog concentrates in the same metabolic pools and has the same
subcellular distribution in the rat heart, where it is found in the
mitochondria. Male Sprague-Dawley rats weighing approximately
300-350 grams, were injected without anesthesia with 40 .mu.Ci of
[.sup.18F]-FCPHA in a total volume of 200 .mu.l. The radiolabeled
FA was injected into the tail vein and the rats subsequently
sacrificed at 5 minutes and 60 minutes post-injection. The organs
were excised and counted in a gamma counter. Accumulation of the FA
in various organs in % DPG (dose per gram) is detailed in Table
1.
TABLE-US-00001 TABLE 1 Accumulation of .sup.18F-FCPHA in Rat Tissue
Tissue % DPG (5 min) % DPG (60 min) Heart 1.55 .+-. 0.66 1.43 .+-.
0.14 Lung 0.47 .+-. 0.12 0.33 .+-. 0.16 Liver 1.34 .+-. 0.26 0.87
.+-. 0.15 Bone 0.16 .+-. 0.03 0.70 .+-. 0.39 Blood 0.064 .+-. 0.008
0.080 .+-. 0.019 Spleen 0.55 .+-. 0.30 0.34 .+-. 0.085 Kidney 0.41
.+-. 0.097 0.34 .+-. 0.08 Adrenal 0.22 .+-. 0.061 0.30 .+-. 0.16
Stomach 0.12 .+-. 0.067 0.15 .+-. 0.038 Gastrointestinal Tract 0.19
.+-. 0.040 0.24 .+-. 0.039 Testes 0.020 .+-. 0.003 0.011 .+-. 0.004
Brain 0.06 .+-. 0.01 0.05 .+-. 0.02 Muscle 0.25 .+-. 0.009 0.097
.+-. 0.075
[0197] Bone accumulation of the fluorinated FA measured
de-fluorination of the compound and its subsequent accumulation. To
confirm imaging resolution, the following ratios were determined at
5 and 60 minutes post-injection (Table 2).
TABLE-US-00002 TABLE 2 Imaging Resolution of .sup.18F-FCPHA Tissue
Ratio Ratio at 5 minutes Ratio at 60 minutes Heart-to-blood 25.8
20.4 Heart-to-lung 3.3 4.6
[0198] Table 3 shows the biodistribution of the .beta.-methyl
analog, [.sup.18F]FBMHA, at 5 and 60 minutes after intravenous
administration in rats (5 per time point). At 5 minutes after
injection, accumulation of radioactivity in the heart was 2.56%
dose per gram, with nearly an equal amount of radioactivity in the
kidneys. Tracer activity in the blood at 5 minutes was 1.02% DPG,
and remained relatively high (0.58% DPG) after 60 minutes. Most of
the radioactivity accumulated in the liver. The heart-to-blood
ratio of 2.6 at 5 minutes did not significantly change after 60
minutes. There was bone accumulation of 0.52% DPG at 5 minutes,
which increased to 2.36% DPG at 60 minutes, indicating extensive
defluorination. Biodistribution of [.sup.18F]FBMHA in rats was
repeated using a 4% Tween-80/saline formulation and similar results
were obtained.
TABLE-US-00003 TABLE 3 Tissue distribution of [.sup.18F]FBMHA in
Sprague-Dawley rats Organ 5 minutes 60 minutes Blood 1.02 .+-. 0.22
0.58 .+-. 0.27 Heart 2.56 .+-. 0.76 1.69 .+-. 0.13 Lung 1.25 .+-.
0.18 0.58 .+-. 0.17 Liver 8.88 .+-. 2.97 4.17 .+-. 1.49 Kidney 2.62
.+-. 0.86 1.66 .+-. 0.61 Muscle 0.66 .+-. 0.22 0.41 .+-. 0.10 Bone
0.52 .+-. 0.16 2.36 .+-. 0.78 Brain 0.25 .+-. 0.07 0.27 .+-.
0.03
[0199] Table 4 represents heart-to-blood ratios in rats 60 minutes
after administration of labeled [.sup.18F]FCPHA compared to other
.beta.-methyl analogs [.sup.18F]FBMHA, [.sup.11C]BMHA, and
[.sup.125I]BMIPP.
TABLE-US-00004 TABLE 4 Heart to blood ratios of various
.beta.-methyl analogs [.sup.18F]FCPHA [.sup.18F]FBMHA
[.sup.11C]BMHA [.sup.125I]BMIPP Ratio 20:1 3:1 10:1 3:1
Example 3
Comparison of the Biodistribution of R,R and S,S Diastereomers with
Racemic Mixtures of .sup.11C or .sup.14C-labeled
3-methyl-heptadecanoic Acid
[0200] The following data compare the biodistribution of
1-[.sup.11C]-3(S)-methylheptadecanoic acid (the `S` isomer) and
1-[.sup.11C]-3(R)-methylheptadecanoic acid (the `R` isomer). Also
compared is the racemic mixture,
1-[.sup.14C]-3(R,S)-methylheptadecanoic acid, labeled with
.sup.14C. Measurement of the % DPE (dose per gram) at 5 minutes and
30 minutes revealed that the `S` isomer has higher heart uptake and
higher heart-to-blood ratios, indicating that the `S` isomer is
preferable than the `R` isomer or the racemic mixture as a
potential heart imaging agent. Biodistribution was performed in six
rats for each compound and time point. The 5-minute distribution
data are contained in Table 5, while the 30-minute data are
contained in Table 6. These unexpected results imply that during
.beta.-oxidation, enoyl-CoA hydratase is stereo-specific and only
recognizes the `S` isomer. Further, stereoisomeric purity is
important for targeting the MFA to heart tissue.
TABLE-US-00005 TABLE 5 Biodistribution of isomers and racemic
mixture of 3-methylheptadecanoic acid and at 5 minutes
post-injection R,S racemic Organ S-isomer R-isomer mixture Blood
0.18 .+-. 0.01 0.30 .+-. 0.04 0.23 .+-. 0.02 Heart 3.44 .+-. 0.54
1.40 .+-. 0.23 2.20 .+-. 0.95 Left Ventricle 4.05 .+-. 0.77 1.50
.+-. 0.50 2.87 .+-. 1.01 Right Ventricle 3.40 .+-. 0.82 0.94 .+-.
0.46 2.05 .+-. 0.54 Heart-to-blood 19.00 4.66 9.56 Left 22.00 5.00
12.48 ventricle-to-blood Right 18.88 3.13 8.90
ventricle-to-blood
TABLE-US-00006 TABLE 6 Biodistribution of isomers and racemic
mixture of 3-methylheptadecanoic acid and at 30 minutes
post-injection R,S racemic Organ S-isomer R-isomer mixture Blood
0.21 .+-. 0.04 0.23 .+-. 0.03 0.20 .+-. 0.02 Heart 3.08 .+-. 0.30
1.07 .+-. 0.16 2.10 .+-. 0.41 Left Ventricle 3.81 .+-. 0.37 1.33
.+-. 0.25 2.81 .+-. 0.35 Right Ventricle 2.82 .+-. 0.46 0.93 .+-.
0.35 2.10 .+-. 0.41 Heart-to-blood 14.66 5.95 10.10 Left 18.14 5.78
12.48 ventricle-to-blood Right 18.88 4.00 10.05
ventricle-to-blood
Example 4
Imaging Studies in Monkeys with [.sup.18F]-labeled
3,4-cyclopropyl-heptadecanoic Acid [.sup.18F]-FCPHA
[0201] Imaging of pig and monkey hearts after injection of
[.sup.18F]FCPHA showed an initial spike of activity corresponding
to blood flow followed by a plateau after 10 minutes (FIG. 3). FIG.
4 represents heart images of a pig at 2-8 minutes post-injection of
[.sup.13N]ammonia (18 mCi), and FIG. 5 displays heart images of the
same pig at 2-8 minutes after intravenous administration of
[.sup.18F]FCPHA (19 mCi). In FIG. 6, the images are a comparison of
[.sup.13N]ammonia (right) and [.sup.18F]FCPHA (left) at 2-8 minutes
in a pig. Pig images had a clearer delineation of the heart muscle
with [.sup.18F]FCPHA compared with that from [.sup.13N]ammonia. The
[.sup.18F]FCPHA late images at 60 minutes exhibited high myocardial
activity retention.
[0202] The biodistribution of [.sup.18F]-FCPHA was also studied in
monkeys. Sixty minutes after intravenous injection of 5 mCi of
[.sup.18F]-FCPHA, the radiolabeled FA analog was visualized by
positron emission tomography (PET) and clearly demonstrates
myocardial trapping in both left and right ventricles (FIG. 7).
FIG. 8 shows transverse heart level slices of a monkey 60 minutes
after administration of [.sup.18F]FCPHA. Blood activity showed fast
clearance in both pig and monkey.
Example 5
Synthesis of Terminally-labeled
[.sup.18F]-fluorophenyl-3,4-cyclopropyl-heptadecanoic Acid
[0203] Based on the aforementioned protocol described in Example 1,
synthesis of this molecule can be achieved by using a different
starting material (such as 10-phenyl-1-decanol). This starting
material, 10-phenyl-1-decanol, was reacted with triphenylphosphine
and dissolved in benzene. A solution of carbon tetrabromide in
benzene was added slowly and the mixture was refluxed for 90
minutes. The reaction mixture was then cooled and filtered, and the
residue washed three times with portions of petroleum ether. The
residue was evaporated to dryness and then stirred with petroleum
ether and left overnight in a freezer. The solution was then
filtered, the resultant residue washed twice with petroleum ether,
and the combined solution evaporated to dryness. This is predicted
to yield an alkyl bromide variant of the starting product, wherein
the bromine atom is substituted for the primary alcohol moiety.
[0204] The brominated compound was subjected to Grignard synthesis
as follows. The brominated compound in ether was injected into
refluxed ether containing magnesium. Reflux continued for 90
minutes under an argon or nitrogen atmosphere. The reaction mixture
was then cooled to room temperature, then subsequently injected
into a .sup.7CO.sub.2 trap and the solution shaken for 5 minutes.
The solution was transferred to a separatory funnel, washed twice
with ether, and combined with 1N HCl. The solution was washed twice
with water, dried using Na.sub.2SO.sub.4 and evaporated. The
resulting compound was predicted to be 17-phenyl-1-heptadecanol.
The following steps after the second PCC oxidation will continue as
described in Example 1. This synthesis yields an analog that
contains a terminal phenyl group and a cyclopropyl moiety at the
3,4-carbon position (see FIG. 9).
[0205] The .sup.18F or .sup.123I radiolabel can be added to the
terminal phenyl moiety by other methods, such as the Schiemann
reaction. For .sup.18F labeling the benzene moiety can undergo
nitration in nitric acid (HNO.sub.3) and sulfuric acid
(H.sub.2SO.sub.4), followed by reduction with Sn and HCl. This
yields a phenyl moiety labeled with an amino group (aniline).
Incubation with NaNO.sub.2 and HCl will convert the amino group to
the diazonium ion. The diazonium salt is the n subjected to
fluorination with [.sup.18F]-HBF.sub.4. This yields a phenyl group
mono-substituted with fluorine. For .sup.123I labeling, the benzene
substituent will undergo nitration in HNO.sub.3 and
H.sub.2SO.sub.4, yielding an aniline group. NaNO.sub.2 and HCl,
followed by iodination with potassium iodide (KI). The iodinated
aryl derivative is radiolabeled by an exchange reaction with
radioiodide in an acid media at high temperatures. Other methods
include preparation of a corresponding tributyl-tin derivative,
followed by electrophilic aromatic radioiodination.
Example 6
Synthesis of endo-[.sup.18F] Fluoro- and [.sup.123I]
Iodo-3,4-cycloproylheptadecanoic Acid Variants
[0206] Syntheses of endo-vinylic radiolabeled variants of FCPHA
were carried out essentially as described in Example 1, but with
modifications. A solution of 6-benzyloxy-1-hexanol was treated with
PCC in methylene chloride at 25.degree. C. The black mixture was
stirred for 2 h and filtered through silica gel. The solvent was
removed and the crude oil was separated on silica gel using
hexane/ethyl acetate (90:10).
[0207] The resulting aldehyde, 6-benzyloxy-1-hexanal, was mixed
with (carbethoxymethylene) triphenylphosphorane in methylene
chloride at 25.degree. C. The mixture was stirred overnight, after
which the solvent was removed and replaced with hexane. Solid
matter was removed by filtration. Chromatography on silica gel,
hexane/ethyl acetate (90:10) yielded the corresponding ethyl
ester.
[0208] Diisobutylaluminum hydride in hexane was added dropwise to
the ethyl ester in methylene chloride at -78.degree. C. The mixture
was allowed to slowly warm to 0.degree. C. and ethyl acetate was
added dropwise, followed by ice. The resultant slurry was acidified
with 10% HCl and extracted with ether. The combined ether extracts
were washed with brine, dried, and solvent was removed.
Chromatography of the crude oil using hexane/ethyl acetate (80:20)
yielded the unsaturated primary alcohol.
[0209] The unsaturated primary alcohol was subjected to
cyclopropanation essentially as described in Charette and
coworkers, as well as Example 1. Dry methylene chloride was cooled
to -25.degree. C. and placed under a nitrogen atmosphere. Diethyl
zinc, followed by 1,2-dimethoxyethane (DME), was added.
Diiodomethane was added dropwise to the solution, while maintaining
the reaction temperature between -25.degree. C. and -10.degree. C.
This solution was then added by double-ended needle, to a solution
containing the unsaturated primary alcohol, dioxaborolane (made
from (+)N,N,N',N'-tetramethyl-L-tartaramide and butylboronic acid),
and 4 .ANG. molecular sieves in methylene chloride under nitrogen
at -40.degree. C. and -30.degree. C. The reaction mixture was
stirred for 2 hours at -25.degree. C., then allowed to warm to
0.degree. C. Saturated ammonium chloride was added to the mixture,
then stirred at RT for 16 hours. Solid matter was removed by
filtration and the layers were separated. The aqueous layer was
extracted with methylene chloride, while organic layers were
pooled, washed with water, and dried. The crude oil was separated
by silica gel chromatography in hexane:ethyl acetate (90:10),
yielding the cyclopropyl alcohol. This reaction was repeated
twice.
[0210] Iodine was added in portions to a solution containing the
cyclopropyl alcohol and triphenylphosphine in dimethylformamide.
The dark red mixture was combined with 10% sodium thiosulfate and
extracted with ether. The combined extract was washed with water,
brine, and the solvent was subsequently removed. Silica gel
chromatography in hexane:ethyl:acetate (95:5) yielded the alkyl
iodide. This alkyl iodide, was combined with sodium cyanide in
dimethyl sulfoxide (DMSO), was heated to 80.degree. C. for 2 hours.
The mixture was poured into water, and was then followed by ether
extraction. The combined extract was washed with water, dried, and
solvent removed. The crude oil was separated by silica gel
chromatography in hexane:ethyl acetate (80:20) and yielded the
corresponding nitrile compound.
[0211] The corresponding nitrile compound was mixed with KOH and
water in ethylene glycol, then heated for 6 h at 170.degree. C.
Once cooled, the mixture was diluted with 10% HCl and extracted
with ether. The combined extract was dried and the solvent removed.
The crude carboxylic acid was treated with diazomethane (made from
N-methyl-N'-nitro-N-nitrosoguanidine and 40% KOH in ether). The
reaction mixture was stirred for 1 h before solvent removal.
Chromatography of the crude oil on silica gel and hexane/ethyl
acetate (80:20) yielded the corresponding methyl ester.
[0212] The methyl ester was hydrogenated using lithium aluminum
hydride, once again yielding a primary alcohol. The primary alcohol
was substituted by addition of an alcohol-protecting group,
tetrahydropyran (THP). Subsequent hydrogenation of the primary
alcohol with 10% palladium on charcoal (Degussa) in methanol,
followed by treatment with N-bromosuccinimide, yielded an alkyl
bromide variant, wherein the bromine group was appended on the
opposite end from the THP protecting group. The alkyl bromide
variant was then subjected to treatment with n-butyllithium in
hexane, in the presence of an alkyne, which appended the alkyne
group opposite from the THP moiety. The corresponding cyclopropyl
alkyne was hydrogenated with tributyl-tin hydride and iodinated
with 12 to yield an endo-vinyl variant.
[0213] The endo-vinyl variant molecule was treated with TosH and
CrO.sub.3 under acidic conditions to facilitate removal of the THP
protecting group and subsequent oxidation to the carboxylic acid.
The corresponding carboxylic acid was then subjected to
substitution of the iodide with tributyl-tin hydride. Radiolabeled
sodium iodide (Na.sup.123I) afforded substitution of the
tributyl-tin moiety with .sup.123I, resulting in
endo-[.sup.123I]-iodo-3,4-cyclopropylheptadecanoic acid.
Alternatively, the tributyl-tin substituted carboxylic acid was
treated with .sup.18F.sub.2 to yield
endo-[.sup.18F]-fluoro-3,4-cyclopropylheptadecanoic acid. A
schematic overview of the synthesis described above is provided in
FIG. 10.
Example 7
Synthesis of exo-[.sup.18F] fluoro- and [.sup.123I]
iodo-3,4-cyclopropylheptadecanoic Acid Variants
[0214] An overview of the partial synthesis of the exo-vinyl
compounds of 3,4-cyclopropyl-heptadecanoic acid is shown in FIG.
12. Synthesis of radiolabeled exo-vinyl variants of
3,4-cyclopropyl-heptadecanoic acid was achieved essentially as
described in Example 1, up until formation of
9-hydroxy-3,4-cyclopropylheptadecanoate methyl ester. This methyl
ester was oxidized to its corresponding ketone with pyridinium
dichromate (PDC) in methylene chloride at 25.degree. C. The
corresponding ketone can then be subjected to two different routes,
depending on the radiolabeled halogen used.
[0215] Vinylic Iodination: Scheme 1
[0216] The following syntheses are based on the Horner-Witting
reaction, with modifications described in Stork and Zhao, 1989.
Tetrahedron Lett. 30: 2173-2174; and McCarthy et al. 1991. J. Am.
Chem. Soc. 113: 7439-7440 (see also FIG. 12). Pre-formed
iodomethyl-triphenylphosphonium iodide, consisting of
triphenylphosphine and the iodinated ylide, was added slowly to a 1
M solution of sodium hexamethyldisilazane in tetrahydrofuran (THF).
After stirring for one minute, the solution was cooled to
-60.degree. C. and hexamethylphosphorictriamide (HMPA) was added,
followed by cooling to -78.degree. C. The ketone compound described
above was added, removed from the cold bath, and stirred for 30
minutes. Hexane was added to the mixture, followed by column
chromatography and NMR analysis. This afforded substitution of the
carbonyl group with the iodinated vinyl group. Subsequent treatment
with tributyl-tin hydride and palladium on carbon yielded
substitution of the vinylic iodine with tributyl-tin and conversion
of the primary ester group to carboxylic acid. Treatment with
radioiodinated sodium iodide (Na .sup.123I) resulted in
exo-[.sup.123I]-iodo-3,4-cyclopropylheptadecanoic acid.
[0217] Vinylic Iodination: Scheme 2
[0218] Likewise, the addition of the radiolabeled iodine can be
directly achieved, thereby eliminating the intervening vinylic
tributyl-tin precursor molecule (FIG. 12). After substitution of
the carbonyl group with the iodinated vinyl group, the molecule can
then be reacted with copper and Na.sup.123I directly, resulting in
exo-[.sup.123I]-iodo-3,4-cyclopropyl-heptadecanoic acid.
[0219] Vinylic Fluorination: Scheme 1
[0220] The vinylic tributyl-tin fatty acid derivative described in
Scheme 1 of vinylic iodination can also be used to directly
fluorinate the modified fatty acid by substitution of
.sup.18F.sub.2 at the vinylic tributyl-tin moiety (FIG. 12). This
yields exo-[.sup.18F] fluoro-3,4-cyclopropyl-heptadecanoic
acid.
[0221] Vinylic Fluorination: Scheme 2
[0222] An alternate synthesis of the vinylic fluorinated variant of
3,4-cyclopropyl-heptadecanoic acid can be achieved from the ketone
compound described above (FIG. 12). A modified version of the
mechanism is from Burton and Greenlimb 1975. J. Org. Chem. 40:
2796-2801; and in Schlosser and Zimmermann 1969. Synthesis 1:
75-76. Fluoromethyltriphenylphosphonium iodide was synthesized in a
round-bottomed flask fitted with a water-cooled reflux condenser.
The flask was charged with triphenylphosphine and fluoroiodomethane
in dry benzene. The resulting solution was refluxed for 64 hr with
moderate stirring. Upon completion of the reaction, the white
insoluble phosphonium salt was collected on a glass funnel, washed
with hot benzene, and dried in a vacuum dessicator for 12 hr.
[0223] A separate flask was equipped with a constant-pressure
addition funnel fitted with a nitrogen inlet, and a water-cooled
reflux condenser topped with a T joint leading to a mineral oil
bubbler. The apparatus was flushed with nitrogen, flame-dried and
allowed to cool to room temperature while de-aeration with nitrogen
was continued. The flask was charged with
fluoromethyltriphenylphosphonium iodide, dry THF, and toluene. The
nitrogen atmosphere was maintained throughout the system. The
slurry of phosphonium salt and solvent was moderately stirred while
cooled in a dry ice-isopropanol slush bath. During the cooling
process, the addition funnel was charged with n-butyllithium in
hexane. Any appreciable amount of hydrolysis of the organolithium
reagent was avoided by a constant sweep of nitrogen over the
addition funnel when the stopper was removed and by transferring
the base via pipette under a dry nitrogen atmosphere. The base was
added dropwise over a 23-minute period. After addition, the
reaction mixture was stirred for 25 minutes at -78.degree. C. At
the end of the 25-minute period, the reaction mixture was withdrawn
under a nitrogen atmosphere and added to ethylene bromide with the
evolution of heat. The reaction was allowed to progress for another
25 minutes at -78.degree. C.
[0224] The corresponding ketone obtained from PDC oxidation of the
methyl ester 9-hydroxy-3,4-cyclopropylheptadecanoate was then
added. The reaction between the ylide and the ketone proceeded for
2 h at -78.degree. C., then allowed to warm to room temperature,
where it was incubated for an additional 1.5 h. The reaction
mixture was then cooled on ice. Potassium tert-butoxide was added
and the mixture was stirred for 2 h at 0.degree. C. The reaction
mixture was centrifuged and decanted, and the precipitate washed
with small portions of THF. The decantates were combined and washed
with saturated aqueous sodium chloride until the aqueous layer was
of neutral pH. The solvent was removed, the dried and filtered
organic material was flash-distilled at reduced pressure, and then
subjected to chromatography and NMR analysis. Base hydrolysis of
the .sup.18F-substituted ester resulted in conversion to the
corresponding carboxylic acid. This yielded
exo-[.sup.18F]-fluoro-3,4-cyclopropylheptadecanoic acid.
Example 8
Synthesis of 4 or 5-membered Ring Substituent Variations of
Heptadecanoic Acid
[0225] Variations on heptadecanoic acid have also been synthesized,
wherein the cyclopropyl substituent is replaced instead with
cyclobutyl or cyclopentyl moieties. Synthesis of these compounds
containing variations of the modified fatty acids described in
Examples 1, 4, 5, and 6 are provided below.
[0226] Synthesis of MFAs with the cyclopropyl moiety was described
in prior examples. However, instead of 6-benzyloxy-1-hexanol, which
was used as starting material for many of the aforementioned MFAs,
cyclic primary alcohols (cyclobutanol or cyclopentanol) can be
substituted with a protective group like THP (tetrahydropyran). The
mono-protected cyclic alcohol is then subjected to
triphenylphosphine in the presence of 12, which resulted in an
alkyl iodide variant, protected at the other end by THP. Addition
of a benzyloxy-alkyne of varying chain length was achieved by
addition of n-butyllithium. Hydrogenation in 10% palladium on
carbon resulted in a cyclobutyl or cyclopentyl-1-nonanol or
heptanol, still protected at one end by THP. The chain length of
the cyclic monoprotected primary alcohol depends on the length of
the benzyloxy-alkyne used. These cyclic mono-protected alcohols
were used in subsequent reactions, described below. FIG. 14
contains a diagram of the partial synthesis of these cyclic
mono-protected alcohols.
[0227] [.sup.18F]-9-fluoro-3,4-cyclobutyl- or
[.sup.18F]-9-fluoro-3,4-cyclopentyl-heptadecanoic Acid
[0228] The cyclic mono-protected nonanol was subjected to PCC
oxidation, yielding the corresponding aldehyde. Under a nitrogen
atmosphere, octyl bromide in dry ether was added to magnesium metal
in ether at a rate as to maintain gentle reflux. After addition was
complete, the reaction mixture was stirred for 1 h and the cyclic
mono-protected nonanal in ether, was added dropwise to the reaction
mixture at room temperature. The mixture was stirred for 4 h, then
poured over ice water, acidified in 10% HCl, and extracted with
ether. The combined extracts were washed with brine, dried, and
solvent removed. Chromatography on silica gel using hexane/ethyl
acetate (80:20) yielded the cyclic mono-protected heptadecanol.
[0229] The cyclic mono-protected heptadecanol was then subjected to
de-protection by treatment with TosH and CrO.sub.3. This yielded
the formation of the corresponding cyclic carboxylic acid. The
cyclic carboxylic acid was combined with methane sulfonyl chloride
and 4-dimethyl aminopyridine (DMAP) in methylene chloride/pyridine
(90:10). The mixture was stirred for 2 h, then washed with 10% HCl
until the aqueous layer was acidic and then washed with 10%
NaHCO.sub.3. The crude oil was chromatographed on silica gel in
methylene chloride/methanol (95:5).
[0230] .sup.18Fluorine in water was added to a vial containing
Kryptofix-222 and K.sub.2CO.sub.3. Water was removed using a
nitrogen stream at 115.degree. C. followed by addition of
acetonitrile. To this vial was the mesylated carboxylic acid in
acetonitrile. The reaction was heated at 120.degree. C. for 10
minutes, which gave the cyclobutyl or cyclopentyl variant of
heptadecanoic acid [.sup.18F]-labeled at the 9-carbon position,
followed by silica Sep-Pak purification in hexane/ethyl acetate
(85:15).
[0231] Terminally-labeled [.sup.18F]-fluorophenyl-3,4-cyclobutyl-
or cyclopentyl-heptadecanoic acid
[0232] Based on the aforementioned protocol described in Example 4,
synthesis of this molecule can be achieved by using a different
starting material (such as 10-phenyl-1-decanol). This starting
material, 10-phenyl-1-decanol, was reacted with triphenylphosphine
and dissolved in benzene. A solution of carbon tetrabromide in
benzene was added slowly and the mixture was refluxed for 90
minutes. The reaction mixture was then cooled and filtered, and the
residue washed three times with portions of petroleum ether. The
residue was evaporated to dryness and then stirred with petroleum
ether and left overnight in a freezer. The solution was then
filtered, the resultant residue washed twice with petroleum ether,
and the combined solution evaporated to dryness. This is predicted
to yield an alkyl bromide variant of the starting product, wherein
the bromine atom is substituted for the primary alcohol moiety.
[0233] The brominated compound was subjected to Grignard synthesis
as follows. The brominated compound in ether was injected into
refluxed ether containing magnesium. Reflux continued for 90
minutes under an argon or nitrogen atmosphere. The reaction mixture
was then cooled to room temperature, then subsequently injected
into a trap containing a cyclobutyl- or cyclopentyl mono-protected
1-heptanol and the solution shaken for 5 minutes. The solution was
transferred to a separatory funnel, washed twice with ether, and
combined with 1N HCl. The solution was washed twice with water,
dried using Na.sub.2SO.sub.4 and evaporated. The resulting
heptadecanol compound was predicted to contain a terminal phenyl
group at one end, with a cyclobutyl or cyclopentyl substituent
followed by the THP protective group.
[0234] The cyclic mono-protected heptadecanol was then subjected to
de-protection by treatment with TosH and CrO.sub.3. This yielded
the formation of the corresponding cyclic carboxylic acid. The
cyclic carboxylic acid was combined with methane sulfonyl chloride
and 4-dimethyl aminopyridine (DMAP) in methylene chloride/pyridine
(90:10). The mixture was stirred for 2 h, then washed with 10% HCl
until the aqueous layer was acidic and then washed with 10%
NaHCO.sub.3. The crude oil was chromatographed on silica gel in
methylene chloride/methanol (95:5).
[0235] The .sup.18F or .sup.123I radiolabel can be added to the
terminal phenyl moiety by other methods, such as the Schiemann
reaction. For .sup.18F labeling the benzene moiety can undergo
nitration in nitric acid (HNO.sub.3) and sulfuric acid
(H.sub.2SO.sub.4), followed by reduction with Sn and HCl. This
yields a phenyl moiety labeled with an amino group (aniline).
Incubation with NaNO.sub.2 and HCl will convert the amino group to
the diazonium ion. The diazonium salt is then subjected to
fluorination with [.sup.18F]-HBF.sub.4. This yields a phenyl group
mono-substituted with fluorine. For 31 labeling, the benzene
substituent will undergo nitration in HNO.sub.3 and
H.sub.2SO.sub.4, yielding an aniline group. NaNO.sub.2 and HCl,
followed by iodination with potassium iodide (KI). The iodinated
aryl derivative is radiolabeled by an exchange reaction with
radioiodide in an acid media at high temperatures. Other methods
include preparation of a corresponding tributyl-tin derivative,
followed by electrophilic aromatic radioiodination.
[0236] Synthesis of Endo-Vinylic Variants of Cyclobutyl or
Cyclopentyl-Substituted Heptadecanoic Acid
[0237] Cyclobutyl and cyclopentyl variants of the endo-vinylic
modified fatty acids described in Example 5 are synthesized
essentially as described, however the starting material, a
cyclobutyl- or cyclopentyl THP mono-protected heptanol, was treated
with N-bromosuccinimide. This yielded an alkyl bromide variant,
wherein the bromine group was appended on the opposite end from the
THP protecting group. The alkyl bromide variant was then subjected
to treatment with n-butyllithium in hexane, in the presence of an
alkyne, which appended the alkyne group opposite from the THP
moiety. The corresponding cyclopropyl alkyne was hydrogenated with
tributyl-tin hydride and iodinated with 12 to yield an endo-vinyl
variant.
[0238] The endo-vinyl variant molecule was treated with TosH and
CrO.sub.3 under acidic conditions to facilitate removal of the THP
protecting group and subsequent oxidation to the carboxylic acid.
The corresponding carboxylic acid was then subjected to
substitution of the iodide with tributyl-tin hydride. Radiolabeled
sodium iodide (Na.sup.131I) afforded substitution of the
tributyl-tin moiety with 1231, resulting in
endo-[.sup.123I]-iodo-3,4-cyclobutyl- or cyclopentyl-heptadecanoic
acid. Alternatively, the tributyl-tin substituted carboxylic acid
was treated with 18F.sub.2 to yield
endo-[.sup.18F]-fluoro-3,4-cyclobutyl- or cyclopentyl-heptadecanoic
acid.
[0239] Synthesis of Exo-Vinylic Variants of Cyclobutyl or
Cyclopentyl-Substituted Heptadecanoic Acid
[0240] The cyclic mono-protected nonanol was subjected to PCC
oxidation, yielding the corresponding aldehyde. Under a nitrogen
atmosphere, octyl bromide in dry ether was added to magnesium metal
in ether at a rate as to maintain gentle reflux. After addition was
complete, the reaction mixture was stirred for 1 h and the cyclic
mono-protected nonanal in ether, was added dropwise to the reaction
mixture at room temperature. The mixture was stirred for 4 h, then
poured over ice water, acidified in 10% HCl, and extracted with
ether. The combined extracts were washed with brine, dried, and
solvent removed. Chromatography on silica gel using hexane/ethyl
acetate (80:20) yielded the cyclic mono-protected heptadecanol.
[0241] The cyclic mono-protected heptadecanol was then subjected to
de-protection by treatment with TosH and CrO.sub.3. This yielded
the formation of the corresponding cyclic carboxylic acid. This
cyclic carboxylic acid derivative was subjected to vinylic
fluorination or iodination as described in Example 6 and modified
from Burton and Greenlimb 1975 J. Org. Chem. 40: 2796-2801; and
also from Schlosser and Zimmermann 1969 Synthesis 1: 75-76.
[0242] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding, it will be apparent to those skilled in the art
that certain changes and modifications can be practiced. Therefore,
the description and examples should not be construed as limiting
the scope of the invention, which is delineated by the appended
claims.
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