(Circulation. 1995;92:1261-1268.)
© 1995 American Heart Association, Inc.
Articles |
Kinetic Analysis of Technetium-99m-Labeled Nitroimidazole (BMS-181321) as a Tracer of Myocardial Hypoxia
Presented in part at the American College of Cardiology, Atlanta, Ga, 1994, and at the Society of Nuclear Medicine, Orlando, Fla, 1994.
From the Yale University–VA Positron Emission Tomography Center (C.K.N., R.S.), Department of Veterans Affairs, West Haven, Conn, and the Division of Nuclear Medicine (C.K.N., A.J.S., B.L.Z., R.S.), Department of Diagnostic Radiology, and the Division of Cardiovascular Medicine (A.J.S., B.L.Z., R.S.), Department of Medicine, Yale University School of Medicine, New Haven, Conn.
Correspondence to Chin K. Ng, PhD, Yale University/VA PET Center, VA Medical Center (115A), 950 Campbell Ave, West Haven, CT 06516.
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Abstract |
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Background Experimental data have indicated that [99mTc]- nitroimidazole (BMS-181321) is preferentially taken up in hypoxic tissue; its kinetics, however, has not been fully investigated. The purpose of this study was to address the relation between perfusate oxygen level and myocardial retention of [99mTc]nitroimidazole.
Methods and Results Bolus injection and constant infusion experiments were performed in Langendorff buffer–perfused rat hearts in normoxic and hypoxic conditions. Data were acquired with a pair of NaI detectors. The initial clearance rate of [99mTc]nitroimidazole was approximately 20 seconds and independent of perfusate oxygen level. The slow clearance rate was greater than 3 hours in all perfusion conditions. The tissue retention of [99mTc]nitroimidazole varied from 0.61±0.14% in normoxic conditions to 5.94±1.16% in the most severe hypoxic conditions. In addition, tissue retention was inversely proportional to perfusate oxygen level in a sigmoidal manner. The constant infusion experiments established that the binding rate at 25% oxygen level (1.94±0.38 mL of perfusate/min-g dry wt) was twofold of that at 40% and sevenfold at 100%. The binding rate of [99mTc]nitroimidazole was independent of the perfusion sequence, suggesting irreversible binding.
Conclusions These data indicate that [99mTc]nitroimidazole may be a useful tracer for the identification of myocardial hypoxia. A sigmoidal relation was demonstrated for the uptake of the tracer, which suggests that a threshold level of hypoxia is necessary for the uptake of the tracer.
Key Words: hypoxia • ischemia
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Currently available noninvasive approaches for the detection of ischemia in patients with coronary artery disease are based on either the identification of flow heterogeneity or regional alterations of myocardial metabolism.1 Studies are commonly performed with [99mTc]sestamibi and thallium-201 in single photon emission computed tomography (SPECT) and [18F]fluorodeoxyglucose, [13N]ammonia, and rubidium-82 in positron emission tomography (PET). These studies using either imaging modality, however, provide only an indirect assessment of regional myocardial ischemia. Since ischemia is tightly coupled to hypoxia, an assessment of tissue oxygen content by tracers extracted readily by the myocardium and retained significantly in direct response to tissue oxygen level may provide a means of positive imaging for the hypoxic myocardium. A class of compounds with high electron affinity, the radiolabeled nitroimidazoles, is therefore under active evaluation for this purpose. These lipophilic compounds diffuse across the cell membrane and undergo reduction in the cytoplasm to form a radical.2 3 4 When oxygen is abundant in the cell, it reacts with the radical anion formed to yield superoxide and noncharged nitroimidazole that then diffuse out of the cell. When intracellular hypoxia is present, the nitroimidazole radical anion is reduced further to form nitrous compounds that combine covalently with cytosolic macromolecules and then are trapped intracellularly. Enhanced retention of nitroimidazoles in hypoxic cells has been demonstrated in dogs5 6 and perfused hearts7 using [18F]fluoromisonidazole and in isolated adult rat myocytes8 and dogs9 using [3H]fluoromisonidazole. For wider clinical applications, [99mTc]nitroimidazole (BMS-181321) has been recently developed to be used as a potential hypoxic marker with the use of SPECT. Preliminary studies in dogs,10 11 perfused hearts,12 and isolated myocytes12 have indicated that [99mTc]nitroimidazole (BMS-181321) may serve as a sensitive marker for hypoxic myocardium. The kinetics of [99mTc]nitroimidazole, however, have not been fully evaluated in the myocardium.
The specific aims of this study were (1) to evaluate the kinetics of [99mTc]nitroimidazole in an isolated perfused heart preparation under a range of hypoxic conditions and (2) to determine the relation between tissue retention and perfusate oxygen level.
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Methods |
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Radionuclide
The structure of [99mTc]nitroimidazole (BMS-181321, Bristol-Myers Squibb) is shown in Fig 1
.
The radiochemical synthesis of BMS-181321 has been described recently
in detail by Linder et al.13 Briefly, a ligand vial was
prepared by dissolving 2.0 mg of propylene amine oxime
(PnAO)-1-(2-nitroimidazole) in 2 mL of saline containing 36 MBq of
[99mTc]TcO4-. Four
milliliters
of saline was added to reconstitute a Squibb Techneplex kit, which
consists of 10 mg pentetate calcium trisodium and 0.5 mg
SnCl2 · 2H2O. The formation of
[99mTc]nitroimidazole was initiated by adding 0.15 mL
of the reconstituted Techneplex into the ligand vial enclosed in a lead
container at room temperature. Radiochemical purity of
[99mTc]nitroimidazole was checked by
thin-layer liquid chromatography (solvent, diethyl
ether) to be >90% after 10 minutes of reaction.
[99mTc]pentetate was prepared with the same procedure
except without the addition of the PnAO-1-(2-nitroimidazole)
ligand.
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Isolated Perfused Rat Heart
Male Sprague-Dawley rats (Harlan)
(weight, 250 to 300
g) were fed ad libitum. The procedures followed for the perfused heart
studies were in accordance with institutional guidelines. The schematic
for the perfusion apparatus and the detection system are
shown in Fig 2. The perfusate buffer was delivered to a
heat and gas exchanger from a reservoir by a roller pump. Hearts were
perfused in Langendorff mode with a constant flow of 10 mL/min without
recirculation in one of the following conditions: (1) normoxia with
buffer saturated by 95% O2 and 5% CO2
(perfusate oxygen level of 100%) and (2) hypoxia with
perfusate oxygen level reduced systemically from 100% to 75%, 50%,
40%, 25%, and 0% by mixing with 95% N2 and 5%
CO2. Heart rate and systolic and diastolic
pressures were also recorded on-line to determine whether the
perfused heart was suitable for the experiment. The perfusate was
Krebs-Henseleit buffer with the following composition (in mmol/L):
NaCl, 119; KCl, 5.9; MgSO4, 7; H2O,
0.53; NaH2PO4, 1.4;
NaHCO3, 25; CaCl2, 2;
H2O, 1.25; glucose, 5; and insulin, 10 mU/mL.
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External Detection of Tissue Radioactivity
Tissue residue
curves of 99mTc-labeled compounds
were measured externally with a pair of NaI detectors as shown in Fig
2
. The proximity of the detectors to the perfused heart was
important
for increasing detection sensitivity and minimizing motion effects.
During acquisition of tissue residue curves, a cylindrical thin film
surrounded the heart to keep it warm. Each NaI detector consisted of a
64-mm crystal (Bicron) encased in a 20-mm-thick cylindrical lead
shield with a 38-mm-diameter collimator hole. Since the whole heart
was in the field of view of the two detectors, total counts over the
heart, which were always less than 40 000 counts/s, were recorded
as a function of time in all studies. The percent dead-time loss
was less than 1% at a count rate of 40 000 counts/s. Tissue residue
curves were acquired on-line with a digital computer (Leading Edge)
at a temporal sampling rate of 1 second per data point for the bolus
injection experiments and 10 seconds per data point for the constant
infusion experiments.
Experimental Protocol
The experimental protocols for both the
bolus injection and the
constant infusion studies are shown in Fig 3. All
studies involved a baseline period of 15 minutes in which hearts were
perfused at the normal oxygen level of 100%.
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Protocol I: Bolus
Injection Studies of
[99mTc]TcO4-,
[99mTc]Pentetate, and
[99mTc]Nitroimidazole
In protocol I (Fig
3A), the gas mixture was switched to either
0%, 25%, 40%, 50%, 75%, or 100% of perfusate oxygen level at 15
minutes before the injection of the radiopharmaceuticals. This
additional 15-minute period allowed the perfused hearts to equilibrate
with the gas mixture in the perfusate. After 30 minutes of perfusion,
1.5 MBq of [99mTc]TcO4-,
[99mTc]- pentetate, or
[99mTc]nitroimidazole was injected over 5 seconds via the
tubing (Fig 2) into the aortic root of the perfused hearts.
Tissue
residue curves then were acquired for 60 minutes, which was required
for the determination of the slow clearance rate. Five hearts each were
performed in experiments studied with
[99mTc]nitroimidazole and three hearts each for other
experiments. The percent yield of the
[99mTc]nitroimidazole synthesis was about 92%, and the
other 8% could be either
[99mTc]TcO4- or
[99mTc]pentetate; thus, the confounding effect of these
two radiolabeled compounds on the kinetics of
[99mTc]nitroimidazole was examined in additional
experiments.
Protocols IIA and IIB: Constant Infusion
Studies of
[99mTc]Nitroimidazole
The accuracy of determining
percentage of tissue
retention using the bolus technique relies heavily on the detection of
a true peak; therefore, a second set of experiments was performed to
examine the applicability of using the constant infusion technique to
estimate the binding rate of [99mTc]nitroimidazole under
various oxygen levels. In addition, the constant infusion experiments
allow the evaluation of [99mTc]nitroimidazole kinetics in
response to changes in oxygen level within the same heart. Two
protocols were used as outlined in Fig 3. Protocol IIA
(n=3 hearts) was
first performed with normoxia followed by hypoxia, and protocol
IIB (n=3 hearts) was performed in the reverse order. In both protocols,
hearts were initially perfused with normal oxygen level for 15 minutes.
Depending on the protocol, hearts then were perfused with a different
oxygen level for an additional period of 15 minutes. After this
equilibration period, the infusion of
[99mTc]nitroimidazole (2.2 MBq/mL) into the aortic root
was initiated at 0.1 mL/min. In protocol IIA (Fig 3B), hearts
were
infused with [99mTc]nitroimidazole for an additional 30
minutes at 100% O2 followed by two successive intervals of
30 minutes and 20 minutes at 40% and 25% oxygen levels, respectively.
In protocol IIB (Fig 3B), hearts were infused with
[99mTc]nitroimidazole for 30 minutes and 40 minutes at
40% and 100% oxygen levels, respectively. To ensure that the perfused
heart would regain most of its contractility after a
hypoxic insult, the experiment with 25% oxygen level was not performed
in protocol IIB. Furthermore, the objective of protocol IIB was to
examine the effect of 100% perfusate oxygen level on the binding
kinetics of [99mTc]nitroimidazole after a period of
hypoxic insult. Since the objective of the constant infusion protocols
was to provide an alternative method to analyze the kinetics of
[99mTc]nitroimidazole, no constant infusion experiments
were performed with
[99mTc]TcO4-
and [99mTc]pentetate. In addition, since
[99mTc]TcO4- was rapidly
cleared
from the vasculature and the right heart chambers, as seen in
"Results," constant infusion experiments with
[99mTc]TcO4- were not
appropriate.
Data Analysis
Tissue Residue Curves of
99mTc-Labeled
Compounds
Tissue time-activity curves obtained after a bolus of
[99mTc]- nitroimidazole and
[99mTc]pentetate were fitted triexponentially. The fast
and slow clearance rates were determined by the first and third
components, respectively. Given the objective of the current study, the
information for the second component was not used. Tissue residue
curves, however, still required three exponential terms for good fit.
Clearance half-time then was estimated by dividing 0.693 by the
clearance rate.14 All tissue time-activity curves were
normalized to their corresponding peak counts to facilitate comparisons
among different experimental groups. Tissue retention was calculated by
averaging the normalized counts between 14 minutes and 16 minutes
(retention 1) or between 58 minutes and 60 minutes (retention 2). The
effect of perfusate oxygen level on tissue retention of
[99mTc]nitroimidazole was described by the modified Hill
equation15 :
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where TR is the fractional tissue retention of [99mTc]nitroimidazole, [O] is the fractional perfusate oxygen level, and a, b, c, and n are constants. To include the observation that tissue retention was not zero at 100% perfusate oxygen level, the Hill equation was modified by the addition of the constant a. Furthermore, tissue retention reached almost a plateau as oxygen level increased beyond 75%; thus, constant a was set at a value of 0.0061, which was the average tissue retention fraction at 100%. Constants b, c, and n were estimated by nonlinear regression using KALEIDAGRAPH 3.0 (Synergy Software). Since the mean values of retention 1 were not statistically different from those of retention 2 for the [99mTc]nitroimidazole experiments, the mean values of retention 1 were used to estimate the parameters.
Estimation of Tissue Binding Rate of
[99mTc]Nitroimidazole
Since the bolus data
indicated that
[99mTc]nitroimidazole exhibited very slow tissue
clearance in all hypoxic conditions, the tracer is effectively trapped
in the myocardium after the initial clearance through the
vasculature. For tracers with irreversible binding kinetics, the linear
slope indicates the binding rate when the tissue residue curve is
normalized to the delivery of the tracer over time.16 The
Patlak approach has been used extensively for the estimation of
myocardial glucose metabolic rate using
[18F]fluorodeoxyglucose and PET.17 Each
tissue residue curve in the current study was scaled to the infusion
dose by counting 1 mL of perfusate in the same detection system. The
curve then represented the distribution volume of
[99mTc]nitroimidazole over time. In other words,
distribution volume describes the equivalent amount of
perfusate containing [99mTc]nitroimidazole
accumulated in the perfused heart at a particular time. The binding
rate (mL of perfusate/min-g dry wt) of
[99mTc]nitroimidazole for each perfusion condition was
estimated by the fitted slope obtained with linear regression. In
protocol IIA (normoxia-hypoxia), the slope was estimated
from 45 minutes to 55 minutes, 70 minutes to 80 minutes, and 90 minutes
to 100 minutes for perfusate oxygen level of 100%, 40%, and 25%,
respectively. In protocol IIB (hypoxia-normoxia), the slope
was estimated from 45 minutes to 55 minutes and from 90 minutes to 100
minutes for perfusate oxygen level of 40% and 100%,
respectively.
Statistical Analysis
All values are expressed as
mean±SD. Dry weight was determined
by placing the whole heart in an oven overnight. Statistical
analysis was performed with use of STATVIEW 4.1
(Abacus Concepts, Inc). P<.05 was considered significant.
The statistical significance of the changes induced by different study
conditions was assessed by one-way ANOVA and Fisher's test for
Tables 1, 2, and 3. The
paired, two-tailed t test was used to determine the
statistical significance between individual values of retention 1 and
retention 2 before being averaged in Table 2, but the mean
values were
tested by unpaired, two-tailed t-test. Since Table 4
was compiled based on Tables 2 and 3, the
unpaired,
two-tailed t test was used to evaluate the statistical
significance between any two groups in this table. The Bonferroni
procedure was used to adjust the critical P values for
multiple tests performed in Tables 2 and
4.18
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Results |
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Tissue Residue Curves of [99mTc]Nitroimidazole
Characteristic tissue residue curves after bolus injection of [99mTc]nitroimidazole for various degrees of hypoxia are shown in Fig 4
. Curves were scaled
to the peak count rate and plotted on a semi–log scale for easy
visualization. Residue curves then were fitted by a triexponential
function, which appeared to sufficiently describe the kinetics in all
perfusion conditions (R>.99). The first exponential term
mainly describes the tracer clearance through the vasculature and the
right heart chambers. As shown in Fig 4, the initial clearance
was
independent of perfusate oxygen level. The clearance half-time of
[99mTc]nitroimidazole for this initial phase was about 20
seconds, with no statistical significance among all perfusion
conditions (Table 1). The second term was required to describe
the
kinetics at the knee portion of the residue curves. The kinetics during
this phase is indicative of nonbound
[99mTc]nitroimidazole equilibrating between the
interstitial space and the intracellular space. This
immediate phase was not part of the objectives in the current study;
thus, its results were not reported. The triexponential function,
nonetheless, was required for the determination of the slow clearance
rate. The tail portion, which reflects the binding kinetics of cellular
[99mTc]nitroimidazole anion after the nitroreduction
process, reached a plateau in less than 10 minutes in all hypoxic
conditions. As shown in Table 1, the clearance half-time for
this
slow phase was about 5 hours independent of the perfusate oxygen level.
The individual values of retention 1 were statistically different from
those of retention 2 before being averaged, but the mean values were
not (Table 2). This indicates that a shorter study time can be
used to
evaluate myocardial hypoxia with the tracer retention because
of the slow washout rate of the reduced forms of
[99mTc]nitroimidazole.
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Kinetic Disparity Between [99mTc]Nitroimidazole,
[99mTc]TcO4-, and
[99mTc]Pentetate
To further evaluate the effect
of perfusate oxygen level on the
kinetics of [99mTc]TcO4- and
[99mTc]pentetate, experiments were performed with these
radiolabeled compounds under two perfusion conditions: perfusate oxygen
levels of 25% and 100%. The fast clearance half-time for
[99mTc]TcO4- was about 5
seconds, which compared with [99mTc]nitroimidazole was 4
times faster in both perfusion conditions (Table 1). As
expected,
[99mTc]TcO4- was not retained
in
the myocardium to any significant degree after 2
minutes (Fig 5) because its negative charge does not
permit the molecule to diffuse across the cell membrane. Subsequently,
data for the slow clearance half-time of
[99mTc]TcO4- was not
available
and therefore was not reported in Table 1. The clearance
half-time
for [99mTc]pentetate was about 10 seconds, which was
about half that for [99mTc]nitroimidazole. Although the
tissue retention fraction of [99mTc]pentetate was almost
twice that of [99mTc]nitroimidazole in normoxic
conditions (Table 2), the retention fraction of
[99mTc]pentetate remained at about 0.9 with perfusate
oxygen level reduced to 25%, while that of
[99mTc]nitroimidazole increased to about 5.0. The higher
retention of [99mTc]pentetate is probably a result of its
ability to bind to the capillary wall and the interstitial
space, since it has the same fast clearance rate as
[99mTc]TcO4-.
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Dependence of Tissue Retention Fraction on Perfusate Oxygen
Level
Since the retention fraction of the tracer potentially can
be estimated by in vivo imaging, it is imperative that a dependence of
tissue retention fraction on perfusate oxygen level be established for
the determination of cellular oxygen level. The reductive bioactivation
of the nitro group on the imidazole ring requires a cellular enzyme
called nitroreductase. Thus, it is reasonable to use the Hill
equation developed from the work of enzyme kinetics to describe
the relation between tissue retention fraction and fractional perfusate
oxygen level. The Hill equation was modified to include a constant of
0.61 to better explain the data that showed an initial plateau. As
shown in Fig 6, the relation is well described, with an
R value of .99. Constants b, c, and n were determined to
be 0.053±0.004, 0.018±0.021, and 7.84±1.94, respectively.
Large
parameter errors were expected because only six groups with
five hearts each were used for the estimations. It is important to note
that cellular oxygen level must be reduced to at least 60% for the
enzyme to be activated.
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Binding Kinetics of [99mTc]Nitroimidazole in
Tissue
Experiments with constant infusion were used to verify the
percentage of tissue retention and the kinetic sensitivity of
[99mTc]nitroimidazole to acute changes in perfusate
oxygen level. Fig 7A demonstrates that the trapping of
the [99mTc]nitroimidazole anion radical, although small,
existed in the normoxic conditions. More importantly, the degree of
trapping increased as perfusate oxygen level was reduced to
40% and 25%, respectively. Fig 7B further substantiates the
results
obtained with the bolus experiments in that the binding of
[99mTc]- nitroimidazole anion radical is essentially
irreversible. The binding remained irreversible even when the
perfusate oxygen level was returned to 100%. The initial
portion of the tissue residue curves in both Fig 7A and
7B probably is
indicative of the filling process of [99mTc]-
nitroimidazole in the ventricular chambers. When the
perfusate oxygen level was returned to normal in protocol IIB (Fig
7B),
the distribution volume showed a slight decrease initially because the
binding rate at normoxic conditions was much smaller than that at
hypoxic conditions. This change was further confounded by smaller
ventricular chambers in the normoxic condition. The binding
rates for various perfusion conditions are summarized in Table
3. For
perfusate oxygen level of 40%, the binding rates were not
significantly different between the two protocols, but the rates were
different for 100%. The mechanism for this difference, however, is not
clear. It is important to note that the ratio of binding rates in any
two perfusion conditions was in the same order as the ratio of
retention fractions obtained from the bolus injection experiments
(Table 4), thus confirming that the tissue residue curves
obtained by
bolus injection indeed consisted of the true peaks. Therefore,
measurements of tissue retention fractions were valid in the bolus
injection experiments.
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Discussion |
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The current study shows that [99mTc]nitroimidazole (BMS-181321), a new lipophilic 99mTc-labeled compound, is preferentially retained in hypoxic myocardium. The nitro group of the compound is responsible for retention of the tracer in the myocardium, while the remaining portion is responsible for the overall lipid solubility of the molecule. The perfused rat heart model with constant flow was chosen for studying the kinetics of [99mTc]nitroimidazole in myocardium because of the ease of inducing global hypoxia in a well-controlled environment. In this model, the effect of hypoxia on [99mTc]nitroimidazole kinetics could be directly evaluated independent of flow. A period of 15 minutes was used to allow adequate oxygen equilibration in the myocardium before the 99mTc-labeled compounds were administered into the aortic root of the perfused heart. The retention of [99mTc]nitroimidazole in myocardium was found to be inversely proportional to perfusate oxygen level in both bolus injection and constant infusion experiments. Furthermore, results from both sets of experiments demonstrated that the cellular binding of [99mTc]nitroimidazole anion radical was essentially irreversible, although a small amount of nonspecific binding was present in perfusion conditions with normal oxygen level. A threshold for cellular binding was established to be 60% perfusate oxygen level in the bolus injection experiments; ie, perfusate oxygen level must be less than 60% in order for the nitro group of [99mTc]nitroimidazole to be reduced significantly and trapped.
Tracer Kinetics of [99mTc]Nitroimidazole in
Myocardium
In rat heart perfused with O2- or
N2-equilibrated cell medium, Rumsey et al12
reported that the washout of [99mTc]nitroimidazole
displayed biphasic kinetics. Our current data obtained from the bolus
injection experiments appeared to display biphasic kinetics, but a
triexponential function was clearly required to describe the kinetic
behavior of [99mTc]nitroimidazole adequately as
determined by the goodness of fit. The difference in kinetics between
Rumsey et al12 and the current study may be due to
differences in experimental protocols. The slow clearance rate of
[99mTc]nitroimidazole was estimated from the bolus
experiments to be about 5 hours regardless of the perfusate oxygen
level. The optimum duration for the determination of mean life for any
counting process with minimum error is of the order of 3 to 5 mean
lives.19 Therefore, the accurate determination of the slow
clearance rate in the current study will require experiments that last
more than 20 hours, an impracticality for isolated heart studies. Since
we chose an experimental duration less than 2 hours, estimation of the
slow clearance rate is expected to have large variability, as evidenced
by results shown in Table 1. On the contrary, the constant
infusion
experiments with [99mTc]- nitroimidazole can
statistically distinguish the tissue binding rate between a perfusate
oxygen level of 25% and 40% (Table 3). It appears that the
estimation
of binding rate with the constant infusion approach is much more
sensitive than the estimation of the clearance half-time for the
slow phase in the bolus injection experiments. As reported by Smith et
al,20 exponential factors and coefficients cannot always
be equated respectively with compartmental transport rate constants or
compartmental sizes. Compartmental analysis of the tissue
residue curves obtained in the bolus injection experiments may provide
accurate estimation on the binding rate constant and the dissociation
rate constant of [99mTc]nitroimidazole in
tissue.
In contrast to [99mTc]TcO4-, tissue kinetics of [99mTc]- nitroimidazole showed a small degree of binding, which was 0.6% of the injected dose, even at normoxic conditions. It is unclear whether or not the binding is related to nonspecific binding of [99mTc]nitroimidazole similar to [99mTc]pentetate, the avidity of the nitroimidazole reductase enzymes for the reduced compound, or a nitro reduction reaction occurring with the intact technetium nitroimidazole. It is conceivable that nitroimidazole is also a substrate for an enzyme that catalyzes the nitroreduction process independent of the oxygen concentration.21 Nonetheless, the level of binding in normoxic conditions was minimal compared with the hypoxic conditions.
By using the multiple indicator–dilution method and the blood-perfused heart preparation, Dahlberg et al22 showed that [99mTc]nitroimidazole had a high initial extraction during both ischemic and hypoxic conditions, followed by a rapid clearance. However, they found that low-flow ischemia significantly increased the net extraction (retention) of [99mTc]nitroimidazole at 15 minutes, whereas hypoxia at normal flow did not alter the net extraction of [99mTc]nitroimidazole. As demonstrated by the current study, tissue retention in hypoxia, which was significantly different from normoxia, could be obtained only when a threshold of perfusate oxygen level was reached. Accordingly, they may not have chosen a level beyond this threshold.
The sigmoidal response provides a much more sensitive control of the retention rate in response to variations in perfusate oxygen level. In other words, once the threshold of 60% oxygen level is reached, any subsequent reduction of oxygen will cause a substantial amount of the [99mTc]nitroimidazole anion to be trapped intracellularly. The sigmoidal response suggests that the cellular nitroreduction process for [99mTc]nitroimidazole probably requires an enzyme that is dependent on oxygen concentration. A second possible mechanism may be the decrease in oxygen concentration is important for the increase in the amount of reduced nitroimidazole and the concentration of reducing equivalents being critical for the rate of nitroreductase reactions. If the sigmoidal relation holds true in the in vivo situation, myocardial oxygen level then can be estimated from the accumulation of tissue radioactivity. Myocardial studies with 18F-labeled fluoromisonidazole in intact dogs indicate that perfusion of less than 40% of normal appeared necessary to increase extraction of tracer, suggesting that this level of ischemia is necessary to create tissue hypoxia.6 This finding further supports the sigmoidal response obtained in the current study.
Tissue retention of [99mTc]nitroimidazole obtained in the bolus injection experiments can be affected easily by the detection of true peaks. Since the clearance of [99mTc]nitroimidazole was found to be very slow, we chose to perform a second set of experiments with the constant infusion technique, which is free from the problem of peak detection. In addition, the constant infusion experiments provide data that reflect the accumulation of [99mTc]nitroimidazole in tissue rather than its clearance from the tissue. The binding rates observed in normoxic conditions were significantly different from each other in the protocols. In protocol IIB, hearts were first perfused with 40% of oxygen level, then with 100%. The data appear to suggest that the initial perfusion with 40% oxygen level caused the [99mTc]nitroimidazole radical anion to occupy some binding sites that were not available for binding when the perfusate oxygen level was changed to 100%. The constant infusion experiments can be used to compare different hypoxic agents in terms of their binding ability.
Considerations in the Experimental Model
There is
considerable disagreement about the adequacy of
oxygenation in isolated hearts perfused with
Krebs-Henseleit buffer. Issues concerning the validity of using the
buffer-perfused hearts for physiological and
biochemical measurements have been addressed extensively by
Opie.23 Furthermore, Leiris and coworkers24
outlined the advantages of using isolated heart preparations in studies
designed to investigate the effect of hypoxia or
ischemia on myocardial cells. Because our major objective for
the current study was to investigate the effect of hypoxia on
the tissue kinetics of [99mTc]nitroimidazole, we
therefore chose the isolated heart preparation perfused with
buffer.
Slight hypoxia in the isolated heart preparation will cause
additional tissue binding of [99mTc]nitroimidazole in the
current study. If the preparation was becoming more hypoxic with time,
the binding rate of [99mTc]nitroimidazole in the
normoxic phase obtained in protocol IIB would have been larger than
that obtained in protocol IIA. At the end of each 2-hour experiment,
the perfused heart plus its water content rarely weighed more than 1.5
g. As shown in Fig 7A, the distribution volume already
increased as
much as 1.2 mL over 30 minutes at 100% perfusate oxygen level. Because
edema merely increases the water space for tracer accumulation, it is
very unlikely that edema is the major cause for the temporal change in
tissue binding kinetics of [99mTc]nitroimidazole in
the constant infusion experiments.
Although we did not measure intracellular oxygen concentration, we allowed a period of 15 minutes for the perfused hearts to equilibrate with the perfusate oxygen concentration. Sarcoplasmic oxygen pressure was found to be nearly identical to the extracellular oxygen pressure in resting cells and only about 2 mm Hg less in rapidly respiring myocytes.25 Since differences of oxygen pressure within the cardiac myocyte are very small, the perfusate oxygen level in the current study should be an adequate indicator of the intracellular oxygen level. Furthermore, measured MVO2 was previously found to be inversely proportional to the perfusate oxygen level in the same perfused rat heart preparation.26 The perfusate oxygen level was probably a suitable index for the indication of the oxygen state in the perfused heart.
Implications for In Vivo Imaging
The current study provides
evidence that
[99mTc]nitroimidazole may potentially serve as an
imaging marker for hypoxic myocardium. Selective retention
of [99mTc]nitroimidazole in hypoxic
myocardium could be enhanced by more than ninefold (Table 2,
5.94/0.61), depending on the severity of cellular oxygen
deprivation. The sigmoidal curve in Fig 6, however, shows
that profound changes in perfusate oxygen levels of 20% to 50% may be
associated with little changes in tracer retention. This steep gradient
may pose a sensitivity limitation in the in vivo studies with
[99mTc]nitroimidazole and SPECT. Preliminary data
studied with [99mTc]nitroimidazole in a canine model
with a severe left anterior descending coronary artery
stenosis demonstrated preferential uptake of the tracer in the
ischemic zone.11 The high uptake and slow
clearance of [99mTc]nitroimidazole in the
liver,10 11 however, will further complicate the
feasibility of using this 99mTc-labeled compound
clinically for the positive identification of ischemic
myocardium. Because the current data suggest that a
threshold exists for the bioactivation of
[99mTc]nitroimidazole in the myocardium, the
limitation of using this radiotracer in low-flow ischemia
is yet to be determined in patients.
It is important to note that the [99mTc]nitroimidazole compound evaluated in the current study is just one member in a family of nitroimidazole compounds. Results from the current study support further investigation to identify which nitroimidazole compound has the appropriate characteristics for in vivo imaging. Many nitroimidazole compounds are being investigated as a radiosensitizer for tumor therapy.27 In addition, preliminary results indicate that [99mTc]nitroimidazole can be used to identify ischemic tissue at risk for infarction during stroke in a cat model.28 Thus, in vivo imaging with radiolabeled nitroimidazoles potentially has a wide range of applications not only in cardiology but also in neurology and oncology.
Conclusions
The initial rapid clearance of
[99mTc]nitroimidazole
is independent of perfusate oxygen level. Approximately 0.6% of the
injected [99mTc]nitroimidazole was retained in the
normoxic myocardium; thus, the nonspecific binding is
minimal. The retention of [99mTc]nitroimidazole is
inversely proportional to the perfusate oxygen level in a sigmoidal
manner. Perfusate oxygen level must be reduced to below a threshold of
60% to have significant trapping of
[99mTc]nitroimidazole. The binding of
[99mTc]- nitroimidazole in the hypoxic
myocardium is essentially irreversible. Furthermore,
binding rate appears to be a more sensitive parameter for
the measurement of change in perfusate oxygen level than the slow
clearance rate.
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Acknowledgments |
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This work was supported in part by a Grant-in-Aid from the Connecticut Affiliate of the American Heart Association to Dr Ng. The authors would like to thank Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ, for providing the materials required to prepare the hypoxia localizing agent 99mTc-labeled TcO(PnAO)-1-(2-nitroimidazole) (BMS-181321).
Received October 12, 1994; revision received February 16, 1995; accepted February 28, 1995.
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