Enhancement of cellular accumulation of lipophilic cationic organometallic compounds by reduction of intramembrane potential

The invention relates to compositions that comprise (1) lipophilic cationic organometallic complexes, particularly hexakis(2-methoxyisobutylisonitrile)technetium(I) complex, and (2) an agent that decreases the intramembrane potential of a living cell. Agents which decrease the intramembrane potential of a living cell include the lipophilic anions, especially tetraphenylborate anion. The invention also relates to methods in which the compositions are administered in vivo and in vitro when it is desirable to obtain enhanced cellular accumulation of lipophilic cationic organometallic complexes. The compositions and methods are useful for diagnosis and treatment, particularly in vivo tissue imaging.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to compositions comprising a lipophilic 
cationic organometallic complex and an agent that decreases the 
intramembrane potential in a cell and methods whereby these compositions 
may be used in vitro and in vivo. The reduction in intramembrane potential 
results in enhancing the cellular accumulation of the lipophilic cationic 
organometallic compounds. 
2. Description of the Background Art 
Hexakis (alkylisonitrile) technetium (I) complexes are a class of low 
valence technetium (.sup.99m Tc) coordination compounds empirically 
designed as clinical myocardial perfusion imaging agents( Jones, A. G. et 
al. Int. J. Nucl. Med. Biol. 11:225-234 (1984), Holman,B. L., et al., J. 
Nucl. Med. 25:1350-1355 (1984), Holman, B. L., et al., ibid 28:13-18 
(1987), Sporn, V., Clin. Nucl. Med. 13:77-81 (1988)). Conceived to be used 
in a manner similar to thallous chloride for the noninvasive evaluation of 
coronary artery disease, the compounds exploit the more favorable emission 
characteristics of .sup.99m Tc for applications in clinical imaging 
(Strauss, H. W., et al., Radiology 160:577-584 (1986), Deutsch, E., et 
al., Science 214:85-86 (1981)). Chemical analysis of these complexes with 
the ground state .sup.99 Tc isotope shows them to be monovalent cations 
with a central Tc(I) core octahedrally surrounded by six identical ligands 
coordinated through the isonitrile carbon. The terminal alkyl groups, when 
bound to the technetium, encase the metal with a sphere of lipophilicity 
(Jones, A. G., et al., Int. J. Nuc. Med. Biol. 11:225-234 (1984), Mousa, 
S. A., et al., J. Nuc. Med. 28:1351-1357 (1987)). 
While the complex has proven highly successful as a clinical flow tracer 
(Holman, B. L., et al., J. Nucl. Med. 25:1350-1355 (1984); Wacker, F. J., 
et al., J. Nucl. Med. 30:301-311 (1989)), evidence has demonstrated a 
component of myocardial localization dependent on tissue viability 
(Piwnica-Worms, D., et al., J. Nucl. Med. 31:464-472 (1990); Rocco, T. P., 
et al., J. Am. Col. Card. 14:1678-1684 (1989); Sinusas, A. J., et al., J. 
Nucl. Med. 30:756 (1989)). 
In the course of investigating mechanisms of cellular retention of these 
agents, studies demonstrated that neither the lipophilic properties nor 
the cationic charge alone were sufficient to characterize the uptake 
properties of these complexes (Piwnica-Worms, D., et al., Invest. Radiol. 
24:25-29 (1989)). The requirement of lipophilicity and cationic charge for 
myocardial localization raised the possibility that their cellular uptake 
and retention mechanisms are in part determined by mitochondrial and 
plasma membrane potentials in a manner analogous to several other known 
permeant cationic probes of membrane potential (Deutsch, C. J., et al., J. 
Cell. Phys. 99:79-93 (1979); Litchtshtein, D., et al., Proc. Nat'l. Acad. 
Sci. U.S.A. 76:650-654 (1979); Bussolati, O., et al., Biochim. et Biophys. 
Acta 854:240-250 (1986); Akerman, K. E., et al., ibid 546:341-347 (1979); 
Johnson, L. V., et al., Proc. Nat'l. Acad. Sci. U.S.A. 77:990-994 (1980); 
Johnson, L. V., et al., J. Cell. Biol. 88:526-535 (1981); Davis, S., et 
al., J. Biol. Chem. 260:13844-13850 (1985)). 
Subsequent studies of cellular uptake of hexakis(methoxyisobutylisonitrile) 
technetium, a member of the isonitrile class of coordination compounds, 
suggested that the uptake of the compound was affected by alterations in 
the plasma and mitochondrial membrane potentials (Delmon-Moingeon, L. I. 
et al., Cancer Res. 50:2198 (1990); Chiu, M. L., et al., J. Nucl. Med.: 
31:1646-1653 (1990)). 
Data from whole organ cardiac preparations also provide indirect evidence 
for this model of cellular uptake. In perfused rabbit heart, oaubain 
(1.5.times.10.sup.-6 M) and hypoxia alter net tissue extraction of Tc-MIBI 
(Meerdink, D. J., et al., J. Nucl. Med. 30:1500-1506 (1989)). In perfused 
rat hearts, metabolic inhibition with sodium cyanide (10 mM) blocks 50% of 
Tc-MIBI accumulation while membrane disruption with Triton X100 (0.5%) 
inhibits 86% of net uptake (Beanlands, R., et al., Circ. 80(S II):545 
(1989)). In sum, the data are consistent with a membrane transport process 
for Tc-MIBI, like other non-metallic lipophilic cations, involving a 
non-carrier-mediated translocation and passive distribution of the agent 
in response to an imposed transmembrane potential. 
Other uptake models for hexakis (alkylisonitrile) technetium complexes have 
been proposed. Simple lipid partitioning was initially thought to be the 
sole mechanism of localization. In this context, lipophilicity was found 
to correlate well with cellular uptake studies and imaging intensity in 
vivo for the more lipophilic agents developed early in this class 
(Piwnica-Worms, D., et al., Invest. Radiol. 24:25-29 (1989)), although 
exceptions to the trend indicated other factors were involved. 
Alternatively, binding of Tc-MIBI (an agent of intermediate lipophilicity) 
to an 8-10 KDalton cytosolic protein has been proposed (Mousa, S. A., et 
al., J. Nucl. Med. 27:P995 (1986)). 
In evaluating the relative merit of these models, a novel prediction of the 
potential-dependent uptake mechanism for Tc-MIBI is the augmentation of 
uptake kinetics by lipophilic anions. In human lymphocytes (Deutsch, C. 
J., et al., J. Cell. Physiol. 99:79-94 (1979)), for example, the kinetics 
of plasma membrane translocation of tetraphenylphosphonium (TPB), another 
well characterized permeant cationic probe of membrane potential, are 
augmented by the presence in the incubation buffer of the lipophilic anion 
tetraphenylborate (TPB). In addition, low concentrations of TPB increase 
uptake kinetics of other lipophilic cations into isolated heart 
mitochondria (Bakeeva, L. E., et al., Biochim. Biophys. Acta 216:13-21 
(1970)), vesicles prepared from E. coli (Altendorf, K., et al., J. Biol. 
Chem. 250:1405-1412 (1975)), and isolated perfused rat liver (Neef, C., et 
al., Biochemical Pharm. 33:3991-4002 (1984)). 
SUMMARY OF THE INVENTION 
The present invention is based upon the inventors' unexpected discovery 
that a decrease in the intramembrane potential will result not only in an 
increase in the rate of uptake but also in an enhanced accumulation level 
of organometallic lipophilic cationic complexes in a cell in which the 
decrease in potential occurs. 
Accordingly, the present invention includes compositions for enhancing the 
intracellular accumulation of lipophilic cationic organometallic 
complexes. 
The present inventors have designed compositions containing technetium 
complexes and agents that produce the decrease in intramembrane potential. 
The exemplified compositions contain a specific 
hexakis(alkylisonitrile)technetium(I) complex and an agent that reduces 
the intramembrane potential in a cell. These agents include, but are not 
limited to, lipophilic anions and dipolar compounds. Preferred embodiments 
are compositions in which the agent is tetraphenylborate ion, 8-anilino-1- 
naphthalene sulfonate ion, or phloretin. Although the preferred 
compositions may include all members of the hexakis(alkylisonitrile) 
technetium (I) class, a more preferred embodiment of the present invention 
comprises compositions containing 
hexakis(methoxyisobutylisonitrile)technetium(I) (Tc-MIBI). 
The present invention also includes methods for enhancing the intracellular 
accumulation of lipophilic cationic organometallic complexes, such as 
hexakis (alkylisonitrile) technetium(I) complexes, by the 
co-administration of a complex and an agent that reduces the intramembrane 
potential of a cell. The cell may be in vivo or in vitro. 
The methods of the present invention that are effective in vivo include, 
but are not restricted to, diagnostic methods in which the organometallic 
complex is formulated with a radioisotope suitable for detection as a 
diagnostic agent and methods of treatment in which the complex is 
comprised of a radioisotope or other component suitable for therapy. 
Methods of the present invention also include in vivo and in vitro cell 
labelling. When performed in vivo, the imaging may be used to detect both 
abnormal and normal cells. In addition to cell labelling, the methods of 
the present invention can be used to image normal body tissue, such as 
whole organs, and abnormal body tissue such as tumors. 
The methods of the present invention use compositions that comprise a 
combination of a lipophilic cationic organometallic complex and an agent 
that decreases the intramembrane potential. In a preferred embodiment, 
methods of the present invention are effected with a composition that 
contains a combination of Tc-MIBI and tetraphenylborate. 
Following the administration of the compositions of the present invention, 
the complex is accumulated in living cells at levels well in excess of the 
levels of accumulation that are found when the complex is administered in 
the absence of the agent. This invention thus enhances the agent's 
potential usefulness as a diagnostic and therapeutic tool.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention relates to lipophilic cationic organometallic 
complexes, preferably compositions comprising 
hexakis(alkylisonitrile)technetium(I) complexes, and an agent that 
decreases the intramembrane potential of a cell and methods whereby these 
compositions may be used in vitro and in vivo. 
In a more preferred embodiment of the present invention the composition 
comprises hexakis(methoxyisobutylisonitrile)technetium(I) (Tc-MIBI) and 
tetraphenylborate ion. Other preferred embodiments include compositions 
comprising Tc-MIBI and either phloretin 
(3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone; 2',4', 
6'-trihydroxy-3-(p-hydroxyphenyl)propriophenone) or 8-anilino-1 
naphthalene sulfonic acid. Further embodiments of the present invention 
include compositions that are combinations of other 
hexakis(alkylisonitrile)technetium complexes and any agent that decreases 
the cellular intramembrane potential. Examples of such agents are 
lipophilic anions other than TPB, such as, 8-anilino-1-naphthalene 
sulfonic acid, phenyl dicarbaundecaborane and trinitophenol and dipolar 
compounds other than phloretin such as phloroacetophenone and 
p-nitrophenol. Other biologically compatible fluorescent dyes such as 
fluorescein derivatives may also be useful. 
Tc-MIBI in particular, but not exclusively, in this class of compounds, 
possesses the unique combination of properties required to be a probe of 
biological membrane potential. This compound is sufficiently lipophilic to 
partition into and through the hydrophobic core of biological membranes, 
but also combines this property with a delocalized cationic charge which 
renders the compound responsive to the plasma and mitochondrial 
transmembrane potentials. This combination of lipophilicity and 
delocalized charge produces an unusual property for these pharmaceuticals. 
Unlike tissue binding of many other pharmaceuticals that depend on highly 
specific binding sites (high affinity receptors), these pharmaceuticals 
have a non-specific uptake mechanism. However, tissue interaction is 
highly specific for those tissues with high plasma membrane potentials, 
high mitochondrial membrane potentials, or high mitochondrial content, or 
combinations of the above. 
A major obstacle of the use of these pharmaceuticals in patients has been 
the low extraction fraction on first pass (Leppo, J. A., et al., Circ. 
Res. 65:632-639 (1989). Because the uptake by tissues is time-dependent, 
any drug delivered via the blood to a tissue may not have enough time to 
be transported into the cells of the target organ (heart, for example) 
before being washed out of that vascular bed and onto other non-target 
tissues (liver, kidney, for example). 
Physiological studies with myocellular preparations have recently indicated 
that the fundamental biophysical mechanism of uptake and retention of 
TcMIBI is both mitochondrial and plasma membrane potential-dependent 
(Piwnica-Worms, D., et al., Circ. (in press); Chiu, M. L., et al., J. 
Nucl. Med. 31:1646-1653 (1990)). 
In cultured chick myocardial cells, for example, depolarizing either plasma 
membrane potentials with high potassium (K.) buffer or mitochondrial 
membrane potentials with the metabolic uncoupler carbonyl 
cyanide-m-chlorphenylhydrazone (CCCP) decreases the unidirectional influx 
and net cellular accumulation of TC-MIBI. Conversely, hyperpolarizing 
mitochondrial membrane potentials with the ionophore nigericin, a K.sup.+ 
/H.sup.+ exchanger, increases net cellular uptake of Tc-MIBI. In 
addition, mitochondrial hyperpolarization by oligomycin-induced inhibition 
of proton influx through the F.sub.1 F.sub.0 -ATP synthase increases 
Tc-MIBI uptake or retention. However, sodium azide in combination with 
oligomycin virtually eliminates net uptake of the agent. 
To further test a proposed model of Tc-MIBI myocellular accumulation and to 
provide insight into a rational approach for augmenting tissue extraction 
of Tc-MIBI in vivo, the response of myocellular transport of Tc-MIBI to 
permeant and impermeant anions as well as various K concentrations were 
evaluated in cultured chick embryo heart cells. It was reasoned that 
understanding the biophysical mechanism of subcellular localization may 
assist experiments into cellular metabolism and furthermore enable this 
agent to be applied to the whole organism and humans as a tracer of tissue 
energetics in vivo. Both uptake kinetics and net accumulation of Tc-MIBI 
were evaluated in the presence of agents that reduce membrane potential. 
The present inventors have discovered that the addition of a compound that 
decreases the intramembrane potential not only increases the kinetics of 
uptake, as might be theoretically expected, but also dramatically 
increases net accumulation of the Tc-MIBI into cells and mitochondria. 
Suitable compounds include, but are not limited to, lipophilic anions, 
exemplified by tetraphenylborate, and dipolar compounds, exemplified by 
phloretin. Reduction of the potential within the membrane allows more easy 
passage of lipophilic cationic organometallic compounds such as Tc-MIBI 
into the target cells. 
Thus, prior to the present invention, the advantageous properties of Tc 
complexes could not be fully advantageously exploited for broad clinical 
use. 
As discussed above, an advantageous property of Tc-MIBI is that the uptake 
by tissues is non-specific. Thus, any living cell (and potentially, any 
tissue type) can retain the molecule. A further advantage is that Tc-MIBI 
has been shown to be safe in humans as a diagnostic pharmaceutical while 
maintaining the unique combination of properties that allow it to respond 
to membrane potential. Conversely, other classes of lipophilic cations or 
fluorescent probes of membrane potential (e.g., rhodamine 123) have been 
shown to be toxic to cells and mitochondria (Bernel, et al. Science 
218:1117-1119 (1982), Emaus, R. K., et al. Biochim. Biophys. Acta 
850:436-448 (1986), Gear, A. R. L. J. Biol. Chem. 249:3628-3637 (1974)). 
These compounds have not been injected into humans. 
One preferred embodiment relates to the source of the isotope. The specific 
activity of the Tc-MIBI complex synsthesized from TcO.sub.4.sup.- 
obtained directly from commercial molybdenum/technetium generators, is 
extremely high. For example, in various embodiments disclosed herein, 
Tc-MIBI was generally synthesized at 1-6.times.1O.sup.8 Ci/mole. By 
comparison, [.sup.3 H] TPP.sup.+, another lipophilic cation, is commonly 
supplied commercially at 5-100 Ci/mole. This provides an opportunity to 
decrease the molar concentration of cation accumulation by the biological 
preparation, yet remain within detectable limits. Since rhodamine 123 and 
TPP.sup.+ have been reported to have toxic effects on mitochondrial 
function at typical loading activities, the high specific activity and 
therefore low concentrations of Tc-MIBI required for biological 
experiments minimize toxic side effects during physiological 
experimentation and clinical imaging with the enhancement process. 
Accordingly, a preferred embodiment of the present invention is the use of 
Tc-MIBI for myocardial perfusion imaging, in humans or veterinary animals, 
by co-administration of Tc-complexes and agents that decrease the 
intramembrane potential. The co-administration enhances accumulation of 
the drug in the target tissue such that the tissue can be more effectively 
imaged in vivo. 
In alternative embodiments, the compositions can be used to image tissues 
other than heart. Examples of tissues that could be imaged are the organs 
normally found in the body and abnormal body tissues such as tumors. Other 
organs and functions that could be imaged with the compositions include 
hepatobiliary function and excretion, metastatic tumor deposits in the 
liver, pulmonary perfusion and pulmonary thromboembolic disease, renal 
perfusion and excretory function, skeletal muscle perfusion and 
abnormalities of both skeletal muscle and myocardial energetics as may 
occur in cardiomyopathies and diseases of mitochondrial dysfunction such 
as mitochondrial cytopathies or "ragged red fiber" disease. This 
composition could also be used for functional tests of cellular or tissue 
energetics in vitro as, for example, in a test of cellular intregity of 
lymphocytes after Indium-IU labelling. 
In another embodiment, the compositions of the present invention may be 
used to treat disease by the enhancement of tissue uptake of therapeutic 
pharmaceuticals. For example, tumor therapy using cytotoxic agents 
directed at tumor mitochondria could be enhanced as could the cellular 
toxicity of chemotherapeutic agents. Along this line, enhanced tumor 
uptake of radiation sensitizing agents could be promoted. Toward these 
ends we disclose embodiments exemplary of the uptake and retention of Tc 
complexes in the presence of such agents. 
In an alternative embodiment of the present invention, lipophilic cationic 
complexes of paramagnetic metal ions such as Gd, Dys, Fe, or Mn can be 
co-administered with agents that decrease the intramembrane potential. 
Complexes of paramagnetic metal ions produce relaxation enhancement of 
tissues placed within a strong magnetic field. Since relaxation 
enhancement is proportional to the local concentration of the paramagnetic 
metal complex, tissue relaxation differences can be augmented by use of 
the present invention during diagnostic tests with magnetic resonance 
imaging technology. 
In another alternative embodiment of the present invention, lipophilic 
cation complexes of rhenium, in particular, but not limited to hexakis 
(akylisonitrile) rhenium(I) complexes, can be co-administered with agents 
that decrease the intramembrane potential. Because rhenium can produce 
ionizing radiation in sufficient local quantities to serve as a 
therapeutic radiopharmaceutical, the present invention can enhance tissue 
accumulation of the agent and improve its potential use in tumor therapy. 
In a specific in vivo embodiment, TPB (3.6.times.10.sup.-7 moles) in 
dimethyl sulfoxide is injected directly into the jugular vein of a rat 
within 30 seconds prior to the injection of Tc-MIBI (75 .mu.Curies; 
approximately 6 pmoles/mCi). 
In a specific in vitro embodiment of the present invention a combination of 
Tc-MIBI and TPB is administered in vitro to spontaneously contractile 
chick ventricular myocardial cells obtained from 10 day old chick embryo 
hearts disaggregated with trypsin. The optimal concentration range of TPB 
in this method is 3.times.10.sup.-6 M to 3.times.10.sup.-5 M. In further 
disclosed embodiments, Tc-MIBI net accumulation in the chick myocardial 
cells is enhanced by the addition of 10.sup.-4 M phloretin or 10.sup.-4 M 
8-anilino-1-naphthalene sulfonic acid. 
In alternative embodiments of the present methods, cells in vitro may be 
exposed to a combination of Tc-MIBI and either phloretin (10.sup.-4 M) or 
8-amilino-1-naphthalene sulfonic acid (10.sup.-4 M). In further 
embodiments, these cells may be exposed to any combination of a 
hexakis(alkylisonitrile)technetium(I) complex and an agent that decreases 
the intramembrane potential. The agents include lipophilic anions and 
dipolar compounds such as described above. 
In further embodiments of the present methods, the preferred and 
alternative compositions may be administered to any cell type in vitro. 
Cell types include prokaryotic and eukaryotic cells that contain membranes 
which undergo a decrease in intramembrane potential in response to the 
administration of the agents of the invention. 
The in vitro methods are useful for imaging a specific cell type. Such 
imaging can be used, for example, to distinguish a specific cell type 
among a mixture of cells. In such methods each cell type in the mixture 
contains membranes that respond to the reducing agent with a specific 
quantitative alteration in membrane potential. Administration of labelled 
Tc complex and a reducing agent will thus result in a mixture of cell 
types wherein each cell type contains a characteristic amount of the 
labelled complex. The cell type can thus be distinguished by detecting and 
quantifying the label. This type of approach may be quantitative or 
qualitative. 
The in vitro methods are also useful for the assay of agents that affect 
the intramembrane potential. The net accumulation of Tc-MIBI may be used 
as a measure of the alteration of potential that occurs in response to the 
exposure of a cell to a given agent. 
Concentrations of TPB, phloretin, and 8-ANS that are useful in vivo and in 
vitro would be approximately 0.5 .mu.M to 3 .mu.M in the solutions 
immediately bathing the cells or tissues. 
By "organometallic is intended, for the purpose of this invention, a metal 
bound through either a covalent or coordinate bond to a carbon-containing 
ligand or chelate. The person of ordinary skill in the art will readily 
recognize such covalently or coordinately bonded metalo-organic compounds. 
These compounds are discussed extensively in Cotton, F. A. and Wilkinson, 
G. Advanced Inorganic Chemistry Third Edition, Interscience Publishers, 
N.Y. (1966). 
By "lipophilic" cation is intended, for the purpose of this invention, a 
cationic complex with an octanol/water partition coefficient greater than 
0.5. 
By the term "treating" is intended the administration to subjects of the 
compositions of the invention for purposes which include prophylaxis, 
amelioration, or cure of disease. 
By the term "administer" is intended any method for introducing the 
compositions of the present invention into a subject. Typical methods 
include, but are not limited to, oral, intranasal, parenteral 
(intravenous, intramuscular, or subcutaneous), or rectal. When 
administration is for the purpose of treatment, administration may be for 
either a "prophylactic" or "therapeutic" purpose. When provided 
prophylactically, the substance is provided in advance of any symptom. The 
prophylactic administration of the substance serves to prevent or 
attenuate any subsequent symptom. When provided therapeutically, the 
substance is provided at (or shortly after) the onset of a symptom. The 
therapeutic administration of the substance serves to attenuate any actual 
symptom. The administration may also be for diagnostic purposes. The term 
"administer" also relates to the application of a substance ex vivo as in 
cell or organ culture. 
By the term "co-administer" is intended that each of at least two 
components be administered during a time frame wherein the respective 
periods of biological activity overlap. Thus the term includes sequential 
as well as coextensive administration of the compounds of the present 
invention. 
By the term "animal" is intended any living creature that contains cells in 
which the intramembrane potential is reduced by the administration of 
agents of this invention. Foremost among such animals are humans; however, 
the invention is not intended to be so-limiting, it being within the 
contemplation of the present invention to apply the compositions of the 
invention to any and all animals which may experience the benefits of the 
application. 
By "intramembrane potential" is meant the mean free energy difference for a 
hydrophobic ion within an aqueous-lipic bilayer that serves as a barrier 
to transmembrane transport of said ion. This potential is to be 
distinguished from the transmembrane potential that results from 
differential ion distribution from the extracellular space to the 
intracytoplasmic space. 
By "decrease" is intended, for the purpose of this invention, a lowering of 
the net positive potential within a biological membrane in a cell. 
By "accumulation" is intended, for the purposes of this invention, the net 
uptake and retention, within a cell, of the organometallic complexes of 
this invention. 
By "enhance" is intended, for the purposes of this invention, an increase 
in accumulation of organometallic complexes in living cells, such that the 
complexes are accumulated to levels that are in excess of those levels 
that are attained in the absence of the enhancing agent. 
By the term "disease" is intended any deviation from or interruption of the 
normal structure or function of any part, organ, or system (or combination 
thereof) of the body that is manifested by a characteristic set of 
symptoms and signs. 
By "label" is intended any atom or compound associated with a lipophilic 
cationic organometallic complex such that the complex can be located and 
quantitated. The association can be by means of covalent bonds, all means 
of non-covalent bonds, and any means of non-covalent attractive forces. 
The label may be intrinsic (found within the molecule per se, such as a 
radioisotopic form of an element) or extrinsic (found attached to the 
molecule as by a covalent bond or associated with the molecule as by 
non-covalent bonds or other non-covalent attractive chemical forces). 
Examples of types of labels which can be used in the present invention 
include, but are not limited to, enzyme labels, radioisotopic labels, 
non-radioactive isotopic labels, fluorescent labels, toxin labels, and 
chemiluminescent labels. 
By "pharmaceutical compound" is intended a chemical entity, whether in the 
solid, liquid, or gaseous phase, which entity may be used on or 
administered to animals, including humans, as an aid in the diagnosis, 
treatment, or prevention of disease or other abnormal condition, for the 
relief of pain or suffering, or to control or improve any physiologic or 
pathologic condition. The term `compound` should be read to include 
synthetic compounds, natural products and macromolecular entities such as 
polypeptides, polynucleotides, or lipids and also small entities such as 
neurotransmitters, ligands, hormones or elemental compounds. The term 
"compound" is meant to refer to that compound whether it is in a crude 
mixture or purified and isolated. The term "pharmaceutical" should be read 
to include any and all compounds that may be used as defined above. 
By "alkyl" is intended, for the purpose of this invention, any functional 
group of the general formula -CR.sub.3 where R can be identical or 
different and include the elements H, C, N, O, S, F, Cl, Br, and I. 
Representative structures include, but are not limited to, substituents 
consisting of --CH.sub.3, --CH.sub.2 CH.sub.3, CH(CH.sub.3).sub.2, 
--C(CH.sub.3).sub.3, --C(CH.sub.3).sub.2 OCH.sub.3, --C(CH.sub.3).sub.2 
COOCH.sub.3, --C(CH3).sub.2 OCOCH.sub.3 --C(CH.sub.3)CONH.sub.2, --C.sub.6 
H.sub.5, --CH.sub.2 (C.sub.6 H.sub.4)OH, or any of their isomeric forms 
having the general composition as the isonitrile radionuclide complexes in 
U.S. Pat. No. 4,452,774 which is incorporated herein by reference. 
EXAMPLES 
EXAMPLE 1 
Experimental Solutions 
Control buffer was a modified Earle's balanced salt solution (MEBSS) with 
the following composition (mN): Na.sup.+, 145; K.sup.+, 5.4; Ca.sup.2, 
1.2; Mg.sup.2+, 0.8; Cl, 152; H.sub.2 PO.sub.4, 0.8; SO.sub.4.sup.2-, 0.8; 
dextrose, 5.6; HEPES, 4.0; and bovine calf serum, 1% (v/v); pH 
78.4.+-.0.05; 37.degree. C. K-methanesulfonate was made by titration of 
methanesulfonic acid with KOH (Piwnica-Worms, D., et al., J. Gen. Physiol. 
81:731-748 (1983)) and replaced NaCl in some solutions by equimolar 
substitution where indicated. Natetraphenylborate, CCCP, bumetanide and 
valinomycin were dissolved into DMSO prior to addition to buffer. DMSO 
alone has no significant affect on contractile activity, action potential 
configuration (Lieberman, M., et al., Dev. Biol. 31:380-403 (1973)) or 
Tc-MIBI uptake kinetics (Piwnica-Worms, D., et al., Circ. (in press)). 
The K concentration of selected buffers was determined by atomic adsorption 
spectrophotometry (Model 3030, Perkin-Elmer, Norwalk, Conn.) as described 
(Piwnica-Worms, D., et al., Circ. (in press)). 
Synthesis of the radiolabeled compound [.sup.99m Tc]MIBI was performed 
using a one-step kit developed at duPont Medical Products, Billerica, 
Mass. The kit reaction vial contains the isonitrile ligand in the form of 
tetrakis (2-methoxy isobutyl isonitrile) Copper (I) tetrafluoroborate (1.0 
mg), a stannous chloride reducing agent (0.075 mg L-cysteine hydrochloride 
(1.0 mg), Sodium Citrate (2.6 mg) and Mannitol (20. mg). The intrinsically 
radiolabelled complex was formed by adding [.sup.99m Tc]TcO.sub.4.sup.- ( 
20-30 mCi, 2-25 pmol/mCi) in 1-2 ml saline (0.15M, NaCl), obtained from a 
commercial molybdenum/technetium generator (duPont Medical Products, 
Billerica, Mass.), to the kit reaction vial, heating at 100.degree. C. for 
15 min, and allowing to cool to room temperature producing an almost 
quantitative yield of the [.sup.99m Tc](MIBI).sub.6.sup.+ complex. Excess 
reducing agent and starting materials were separated from the 
radiolabelled component as follows: the contents of the reaction vial were 
loaded via syringe onto a reversed phase Sep-Pak cartridge (C-18, Waters 
Assoc., Milford, Mass.) pre-wet with ethanol (3 ml, 90%) followed by 
distilled water (5 ml). Hydrophilic impurities were eluted from the 
cartridge by washing with saline (10 ml, 0.15M) and the desired Tc-MIBI 
collected by elution with ethanol/saline (2 ml; 9:1, v:v). Final total 
.sup.99m Tc activity in the 2 ml effluent (stock) was assayed in a 
standard dose calibrator (CRC-12, Capintec, Ramsey, N.J.). Radiochemical 
purity was found to be greater than 97% by thin layer chromatography 
(aluminum oxide plates, J. T. Baker, Phillipsburg, N.J.) using ethanol 
(absolute) as the mobile phase. 
Statistics 
Values are presented as mean .+-. SEM. Statistical significance was 
determined by the two-tailed unpaired Student's t test where indicated in 
the text (Wallenstein, S., et al., Circ. Res. 47:1-9 (1980)). 
EXAMPLE 2 
Tissue Culture of Chick Myocardial Cells 
Monolayers of spontaneously contractile chick ventricular myocardial cells 
were obtained from disaggregated 10-day old chick embryo hearts by a 
slight modification of previously published methods (Horres, C. R., et 
al., In Pinson, A. (ed.), The Heart Cell in Culture, Boca Raton, CRC 
Press, pp. 77-108 (1987)). Hearts were trimmed of connective tissue and 
atria, finely minced and serially exposed to 0.024% (w/v) trypsin in 
Ca.sup.2+ - and Mg.sup.2+ - free Earle's salt solution for 7 minutes at 
37.degree. C. Gentle trituration and agitation on an orbital shaker bath 
aided disaggregation. Cells released from the first exposure were 
discarded and cells aspirated from the next four exposures were then added 
to an equal Volume of trypsin deactivating solution consisting of ice-cold 
culture medium. Cells were centrifuged at 400 g for 10 minutes, 
resuspended and combined in culture medium, counted with a hemocytometer 
and diluted to yield a suspension of 5.times.10.sup.5 cells/ml. 12 ml of 
suspension were incubated in 100 mm plastic culture dishes containing 7 
coverslips (25 mm diameter) placed on the bottom of each dish to serve as 
substrate for cell growth. Cells were maintained in a humidified 
atmosphere of 5% CO.sub.2 /95% air for 3-4 days yielding a confluent layer 
of spontaneously contractile myocytes on each coverslip. 
EXAMPLE 3 
Radiotracer Uptake Methods 
Radioactive uptake methods have been described in detail (Piwnica-Worms, 
D., et al, Circ. (in press). Briefly, coverslips with confluent cells were 
removed from culture media and pre-equilibrated for 40-60 seconds in MEBSS 
buffer. Uptake and retention experiments were initiated by immersion of 
coverslips in 60 mm glass Pyrex dishes containing loading solution 
consisting of buffer with 0.1-0.6 nM [.sup.99m Tc-MIBI] (0.01-0.4 
Ci/nmole; 25-100 uCi/ml). Preparations were removed at various times and 
rinsed three times in 25 ml volumes of ice-cold (2.degree. C.) 
isotope-free buffer for 8 seconds each to clear extracellular spaces. 
Preparations and aliquots of the loading buffer and stock solutions were 
counted in a well-type sodium iodide gamma counter after which cell 
protein on each coverslip was extracted in 1% sodium dodecylsulfate with 
10 mM sodium borate and assayed by the method of Lowry (Lowry, O. H., et 
al., J. Biol. Chem. 193:265-275 (1951)). Tc-MIBI binding to glass 
coverslips without cells was used as an estimate of non-specific adhesion 
to the substrate (&lt;% of total activity obtained with cellular 
preparations); this value was subtracted from total uptake determinations 
to derive the cell-associated counts. Use of generator equilibrium 
equations (Lamson, M. L., et al., J. Nucl. Med. 16:639-641 (1975)) allowed 
calculation of absolute moles of Tc-MIBI in solutions and preparations. 
Results were therefore expressed as fmol cellular Tc-MIBI/mg protein per 
nM extracellular Tc-MIBI concentration. Division of this value by the cell 
water space (6.9 ul/mg protein; Chiu, M. L., et al., J. Nucl. Med. 
(31:1646-1653 (1990)) yields a nominal intracellular/extracellular Tc-MIBI 
accumulation ratio neglecting subcellular compartmentation of the agent. 
EXAMPLE 4 
Effect of TPB on Net Accumulation of Tc-MIBI in Cultured Chick Heart Cells 
Myocytes incubated in MEBSS containing Tc-MIBI (0.5 nM) accumulated the 
lipophilic cation to an apparent equilibrium. A low concentration of 
tetraphenylborate (TPB, 10.sup.-9 M) minimally increased the rate of 
Tc-MIBI accumulation, but did not significantly affect final equilibrium 
content (P&gt;0.5). However, at concentrations equal or greater than 
5.times.10.sup.-7 M, TPB increased both maximal accumulation and the 
uptake kinetics of Tc-MIBI (FIG. 1). In 10.sup.-5 M TPB, peak accumulation 
of Tc-MIBI was 4-fold greater than control (P&lt;0.001) and occurred within 
10-20 minutes, 3-fold faster than control. At these high concentrations of 
TPB, accumulation of Tc-MIBI was not constant over time, but rather 
declined after achieving a transient maximum value. A concentration-effect 
curve for TPB enhancement of myocellular uptake of Tc-MIBI is shown in 
FIG. 2. TPB increased myocellular uptake of Tc-MIBI in a 
concentration-dependent manner up to 3.times.10.sup.-5 M (half-maximal 
concentration .sup..about. 3.times.10.sup.-6 M); higher concentrations of 
the lipohpilic anion markedly reduced net cellular accumulation of 
Tc-MIBI. 
EXAMPLE 5 
Effect of MSA on Tc-MIBI Net Accumulation 
As opposed to TPB, relatively high concentrations of the impermeant anion 
methanesulfonate (MSA; 5.4 mM) (Piwnica-Worms, D., et al., J. Gen. 
Physiol. 81:731-748 (1983)) did not significantly alter uptake kinetics or 
equilibrium content of Tc-MIBI (FIG. 3). Furthermore, 5.4 mM MSA did not 
interfere with the TPB-induced enhancement of Tc-MIBI accumulation (FIG. 
3). Even 130 mM MSA had little effect on Tc-MIBI uptake in the presence of 
TPB (10.sup.-5 M) and 130 mM K. (130 mM KCl buffer + bumetamide [10.sup.-5 
M]: 63.7.+-.3.2 fmol Tc-MIBI/mg protein per nM vs. 130 mM K-MSA buffer: 
79.3.+-.1.1; n=3; P=0.01). 
EXAMPLE 6 
Influence of TPB on Membrane Potential-Independent Accumulation of Tc-MIBI 
To determine whether TPB was increasing membrane partitioning (adsorption) 
of Tc-MIBI or enhancing a potential-dependent component of net uptake of 
Tc-MIBI, cells were exposed to various sequential combinations of Tc-MIBI, 
TPB and the protonophore CCCP. As shown in FIG. 4, TPB (10.sup.-5 M) 
increased myocyte content of Tc-MIBI to similar values above control 
whether added to the buffer at time zero or subsequent to the attainment 
of a Tc-MIBI plateau. The time of onset for TPB enhancement of Tc-MIBI was 
rapid. Augmentation of Tc-MIBI myocellular kinetics could be detected as 
early as 5 seconds into a pre-incubation period with TPB (control: 
1.23.+-.0.15 fmol Tc-MIBI/mg protein per nM; +10.sup.-7 M TPB: 
3.37.+-.0.57; +5.times.10.sup.-6 M TPB: 20.6.+-.0.43; 2 min uptakes with 
n=3-4 each). Depolarizing mitochondiral membrane potentials with CCCP (5 
uM) caused the rapid and near complete release of myocellular Tc-MIBI both 
in the presence and absence of TPB (P&lt;0.001). The CCCP-insensitive 
retention of Tc-MIBI was slightly higher in TPB-treated cells. This could 
reflect either increased lipid partitioning of Tc-MIBI in the presence of 
TPB or differential effects of TPB on membrane potentials under these 
conditions. 
To further explore the influence of TPB on any potential-independent 
accumulation of Tc-MIBI in myocytes, preparations were exposed to 130 mM 
K..degree. 20 mM Cl.sub.o buffer (Table 1) . Intracellular potassium 
(K.sub.i) in these preparations has been previously determined to be 130 
mM (Piwnica-Worms, D., et al., Circ. (in press)), therefore equalizing the 
transmembrane K concentration with this buffer produces an isovolumic 
depolarization of plasma membrane potential to nearly zero millivolts 
(Horres, C. R., et al., Am. J. Physiol. (Cell) 236:C163-C170 (1979)). The 
residual uptake of Tc-MIBI in high K.sub.o buffer could be attributed to 
intact mitochondiral membrane potentials since collapsing mitochondrial 
potentials with valinomycin (lug/ml) eliminated net accumulation of 
Tc-MIBI (Table 1). TPB (1O.sup.-5 M) increased myocellular content of 
Tc-MIBI in high K.sub.o buffer, but again, this augmentation was 
completely valinomycin-sensitive. Equilibration of intracellular and 
extracellular spaces occurred in the presence of high K buffer plus 
valinomycin under all conditions (in/out ration .sup..about. 1; P=NS) 
indicating the lack of any siginificant potential-independent membrane 
binding of Tc-MIBI. 
TABLE 1 
______________________________________ 
Effect of Valinomycin on Tetraphenylborate 
Enhancement of Tc-MIBI Accumulation in High K.sub.o Buffer 
Tc-MIBI Net Uptake 
(fmol/mg Tc-MIBI.sub.i / 
Buffer protein/nM.sub.o) Tc-MIBI.sub.o 
______________________________________ 
130 K.sub.o 
65.3 .+-. 5.3 (n = 3) 9.5 
130 K.sub.o + TPB 
.sup.+ 93.9 .+-. 4.4.sup. 
(n = 3) 13.6 
130 K.sub.o + val 
*6.2 .+-. 0.3 (n = 3) 0.9 
130 K.sub.o + TPB+ 
*7.6 .+-. 0.8 (n = 3) 1.1 
val 
______________________________________ 
Preparations were incubated in high K.sub.o low Cl.sub.o loading buffer +/- 
valinomycin (1 ug/ml) for 20 minutes in the presence of TPB (10.sup.-5 M) 
or for 40 minutes in its absence to determine peak accumulation of Tc-MIBI 
under each condition. Values are the mean .+-. SEM. Tc-MIBI in/out ratios 
were obtained by dividing net uptake values by the cell water space of 6.9 
ul/ug protein (Chiu, M. L., et al., J. Nucl. 31:1646-1653 (1990)). 
+P&lt;0.01; * P&lt;0.001 compared to net uptake in 130 mM K.sub.o buffer. 
EXAMPLE 7 
K.sub.o -Dependence of Tc-MIBI Net Uptake in the Absence and Presence of 
TPB 
The myocellular plasma membrane potential is primarily a K diffusion 
potential for K.sub.o &gt;10 mM (Horres, C. R., et al., Am. J. Physiol. 
(Cell) 236:C163-C170 (1979)); consistent with this, we observed a strong 
dependence of Tc-MIBI net uptake on K.sub.o (FIG. 5). TPB increased 
Tc-MIBI net accumulation at all K.sub.o concentrations and the K.sub.o 
-dependence of Tc-MIBI uptake was preserved. Possible K.sub.o -induced 
cell volume changes could not have significantly contributed to net uptake 
of Tc-MIBI; no differences were found between use of low [Cl].sub.o (K-MSA 
substitution for NaCl) or use of bumetanide (10.sup.-5 M) (an inhibitor of 
the volume-responsive Na+K+2Cl co-transporter (26) during KCl substitution 
for NaCl) to maintain cells isovolumic in various K.sub.o buffers (data 
not shown). Nominal intracellular/extracellular accumulation ratios, 
calculated by dividing the Tc-MIBI content values in FIG. 5 by the 
previously determined cell water of 6.9 ul/mg protein (Chiu, M. L., et 
al., J. Nucl. 31:1646-1653(1990)), were greater than those expected from 
the K.sub.i /K.sub.o ratios in the absence of TPB; this effect was even 
more pronounced in the presence of TBP. This provided further evidence for 
enhanced subcellular compartmentation of Tc-MIBI by TPB. 
EXAMPLE 8 
Effect of TPB on the K.sub.o -Dependence of Tc-MIBI Influx Rates 
Unidirection influx of Tc-MIBI into heart cells determined by 2 min uptake 
values was strongly K.sub.o -dependent, (FIG. 6B). Control experiments 
indicated that complete onset of the valinomycin-induced depolarization of 
mitochondrial potentials required approximately a 3 min pre-incubation 
period (data not shown). Therefore, preparations were pre-treated in 
valinomycin (1 ug/ml) for 5 minutes prior to determination of Tc-MIBI 
influx; use of the Goldman flux equation (Restrepo, D., et al., J. Gen. 
Physiol. 92:489-507 (1988)) allowed estimation of the plasma membrane 
potential as a function of K.sub.o (FIG. 6C). As can be seen, Tc-MIBI 
approximated a Nernstian probe of plasma membrane potential under these 
conditions (slope=-67 mV/decade; r=-0.99). Whereas Tc-MIBI influx in the 
absence of TPB was only modestly valinomycin-sensitive (FIG. 6A), Tc-MIBI 
influx in its presence was highly valinomycin-sensitive (FIG. 6B), 
implying enhanced mitochondrial uptake of Tc-MIBI by TPB. 
EXAMPLE 9 
Effect of Dipolar Compounds on Net Uptake of Tc-MIBI 
The effect, on Tc-MIBI accumulation, of dipolar compounds that decrease the 
intramembrane potential was investigated in cultured chick myocardial 
cells that were prepared by the disaggregation of 10 day old embryos. The 
time course and dose response of phloretin is shown in FIG. 7. At a 
concentration of 10.sup.-4 M phloretin, within 30 minutes of 
administration, the net uptake of Tc-MIBI is enhanced five-fold above the 
control. FIG. 8 shows the results of several experiments in which 30 
minute uptake levels of Tc-MIBI were measured. The uptake was measured in 
the presence of 10.sup.-4 M and 10.sup.-5 M phloretin with or without 
10.sup.-5 M TPB. In the presence of 10.sup.-4 M phloretin, the net 
retention of Tc-MIBI ranges from approximately 2.5-3.5-fold above the 
control levels. 
FIG. 9 shows the net uptake of Tc-MIBI in response to dose and time of 
incubation with 8-anilino-1-naphthalene sulfonic acid. At a concentration 
of 10.sup.-4 M and within 30 minutes, the net uptake of Tc-MIBI is 
enhanced 25% over the control. 
EXAMPLE 10 
Effect of TPB on Tissue Uptake of Tc-MIBI in Animals 
Table 2 shows the levels of uptake of Tc-MIBI in heart, blood, liver, 
kidney and lung of live rats in which TPB and Tc-MIBI were 
co-administered. The results are expressed as percent injected dose per 
gram of tissue. For liver, kidney, lung and blood there was a significant 
increase in Tc-MIBI accumulation within the tissues. For all tissues there 
was a 50% increase in tissue retention of Tc-MIBI from 6.04 to 9.15% ID 
per gram of total tissue. For this series of experiments, 50 microliters 
of dimethylsulfoxide lacking or containing 7.2 mM TPB were injected 
directly into the jugular vein of anesthetized rats immediately prior to 
the injection of Tc-MIBI (150 microliters; 100 microCuries per 200 
microliters). 
After 15 minutes, the animal was sacrificed by thoracotomy, organs 
extracted and placed in test tubes and counted for radioactivity in a 
standard well-type gamma counter. Tissue wet weights were obtained by 
subtracting fare weights from total weight of test tubes plus tissue. 
Results are expressed as mean .+-.SEM of three determinations each. 
TABLE 2 
______________________________________ 
EFFECT OF TETRAPHENYLBORATE ON THE 
BIODISTRIBUTION OF Tc-MIBI IN RATS 
% ID/gm 
Organ -TPB +TPB 
______________________________________ 
Heart 1.92 .+-. 0.51 
1.45 .+-. 0.11 
Liver 0.25 .+-. 0.15 
0.76 .+-. 0.40 
Blood 0.04 .+-. 0.01 
0.06 .+-. 0.01 
Kidney 3.38 .+-. 1.09 
5.81 .+-. 0.74 
Lung 0.45 .+-. 0.09 
1.07 .+-. 0.15 
______________________________________ 
EXAMPLE 11 
Physiological Effects of Modest TPB Concentrations 
Moderate concentrations of TPB produced a significant increase in final 
myocellular content of tracer Tc-MIBI in addition to an increase in 
Tc-MIBI kinetics. While not intending to be held to this interpretation, 
this augmented content of tracer Tc-MIBI appeared to be localized to the 
mitochondria. Evidence in support of this included: 1) The nominal 
myocellular accumulation ratio for equilibrium uptake of Tc-MIBI in the 
presence of TPB exceeded expectations for the plasma membrane potential 
(based upon a Nernstian distribution of the transmembrane K gradient), a 
finding consistent with enhanced compartmentalization of Tc-MIBI within 
mitochondria. 2) The majority of the TPB-enhanced net uptake of Tc-MIBI 
was releasable by the mitochondrial uncoupler CCCP. However, the residual 
CCCP-insensitive Tc-MIBI content, representing only 13% of the 
TPB-enhanced Tc-MIBI content, was slightly higher than the control 
residual. This suggests an increase in a component of 
potential-independent binding of tracer Tc-MIBI in the presence of TPB or 
TPB-induced changes in plasma membrane potentials. 3) However, virtually 
eliminating any contribution from the plasma membrane potential by 
incubating cells in 130 mM K.sub.o buffer (Piwnica-Worms, D., et al., Am. 
J. Physiol. (Cell ) 249:C337-C344 (1985)) and collapsing mitochondrial 
potentials with valinomycin caused equilibration of intracellular and 
extracellular Tc-MIBI in both the presence and absence of TPB. Thus, there 
was no evidence of TPB-induced potential-independent membrane partitioning 
of tracer Tc-MIBI in myocytes under these conditions with moderate 
concentrations of TPB. (The membrane binding saturation of carrier-added 
Tc-MIBI discussed below occurs at 10.sup.6 -fold higher concentrations.) 
Therefore, in sum, the plateau data indicated enhancement of mitochondrial 
localization of tracer Tc-MIBI in the presence of TPB. 
It therefore follows that in the absence of TPB, Tc-MIBI does not 
completely equilibrate with the mitochondrial membrane potential and final 
myocyte Tc-MIBI content under control conditions may have represented only 
a quasi-equilibrium. In accord with the general model of hydrophobic ion 
transport discussed earlier, this may suggest that the mitochondrial 
intramembranous energy barrier is sufficiently positive to inhibit the 
ability of Tc-MIBI to attain maximum distribution ratios. 
Toxicity 
High TPB concentrations (10.sup.-4 M) produced relatively less enhancement 
of Tc-MIBI accumulation compared to moderate TPB concentrations suggesting 
cellular toxicity at high concentrations of the lipophilic anion. These 
data are compatible with the previously reported competitive displacement 
of tracer .sup.99m Tc-MIBI by carrier .sup.99 Tc-MIBI at a half-maximal 
molar ratio (apparent K.sub.D) of 7.times.10.sup.-5 (Piwnica-Worms, D., et 
al., J. Nucl. Med. 31:464-472 (1990)), a result consistent with cellular 
toxicity at very high carrier-added concentrations of the lipophilic 
cation. Therefore, results from cultured heart cells may have demonstrated 
onset of binding saturation (and membrane disruption) for very high 
concentrations of these hydrophobic ions and are consistent with reports 
of electrostatic saturation of both TPB binding to egg phosphatidylcholine 
vesicles at 6.times.10.sup.-4 M (Flewelling, R. F., et al., Biophys. J. 
49:531-540 (1986)) and TPB binding to bacterial phosphatidylethanolamine 
bilayers at concentrations exceeding 10.sup.-6 M (Anderson, O. S., et al., 
Biophys. J. 21:35-70 (1978)). 
Quantitative Evaluation of Membrane Potential 
The dominance of net cellular accumulation of Tc-MIBI on mitochrondrial 
membrane potential stands in contrast to unidirectional cellular influx of 
Tc-MIBI: estimates of unidirectional influx rates of Tc-MIBI were 
dominated by the plasma membrane potential. Use of the Goldman flux 
equation to analyze the K.sub.o -dependence of influx in control buffer 
resulted in overestimation of an ideal Nernstian slope. From the 
perspective of quantitatively evaluating Tc-MIBI as a probe of plasma 
membrane potential, the valinomycin sensitivity of this overestimation 
indicated the small, but detectable, contribution of mitochondria to the 
control influx data. However, in the presence of valinomycin, Tc-MIBI 
demonstrated close to ideal Nernstian behavior. 
Clinical Consequences 
These data have implications for the initial biodistribution in vivo of 
intravenous bolus injections of Tc-MIBI. Initial tissue uptake in vivo, 
simulated in part by the unidirectional influx experiments in vitro, may 
be relatively more influenced by plasma membrane potentials compared to 
mitochondrial potentials. Therefore, Tc-MIBI, although a flow tracer, may 
also be a viability agent responsive to tissue energetics reflected in the 
plasma membrane potentials. 
Having now generally described this invention, it will become readily 
apparent to those skilled in the art that many changes and modifications 
can be made thereto without affecting the spirit or scope thereof.