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Medical Patent Abstract
Methods and compositions for medical imaging, evaluating intracellular
processes and components, radiotherapy of intracellular targets,
and drug delivery by the use of novel cell membrane-permeant peptide
conjugate coordination and covalent complexes having target cell
specificity are provided. Kits for conjugating radionuclides and
other metals to peptide coordination complexes are also provided.
Medical Patent Claims
What is claimed is:
1. A compound, comprising: a cell membrane-permeant peptide consisting
of a peptide sequence selected from the group consisting of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:
35, and SEQ ID NO: 36; a diagnostic or pharmaceutically active substance;
and a non-functional linker moiety linking said peptide and said
diagnostic or pharmaceutically active substance; wherein said cell
membrane-permeant peptide comprises at least one D-amino acid, or
a pharmaceutically acceptable salt of said compound.
2. The compound of claim 1, wherein the majority of the amino acids
comprising said cell membrane-permeant peptide are D-amino acids.
3. The compound of claim 2, wherein said cell membrane-permeant
peptide is comprised entirely of D-amino acids.
4. The compound of claim 1 wherein said cell membrane-permeant
peptide is of SEQ ID NO: 6.
5. The compound of claim 1 wherein said non-functional linker moiety
is selected from the group consisting of amino hexanoic acid, glycine,
alanine, peptide chains of nonpolar amino acids, and hydrocarbon
chains.
6. The compound of claim 1 wherein said diagnostic substance is
selected from the group consisting of a radionuclide, a relaxivity
metal, a fluorochrome, a dye, and an enzyme substrate.
7. The compound of claim 6, wherein said radionuclide or relaxivity
metal is coordinated to a chelation ligand linked to said non-functional
linker moiety.
8. The compound of claim 7, wherein said chelation ligand is selected
from the group consisting of DTPA, EDTA, and DOTA.
9. The compound of claim 8, wherein said radionuclide is a radioactive
isotope of a metal selected from the group consisting Tc, Ru, In,
Ga, Co, Pt, Fe, Os, Ir, W, Re, Cr, Mo, Mn, Ni, Rh, Pd, Nb, Cu, and
Ta.
10. The compound of claim 9, wherein said relaxivity metal is a
paramagnetic isotope of a metal selected from the group consisting
of Mn, Cr, Fe, Gd, Eu, Dy, Ho, Cu, Co, Ni, Sm, Tb, Er, Tm, and Yb.
11. A composition, comprising a compound comprising: a cell membrane-permeant
peptide consisting of a peptide sequence selected from the group
consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID
NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36; a diagnostic or pharmaceutically
active substance; and a non-functional linker moiety linking said
peptide and said diagnostic or pharmaceutically active substance;
wherein said compound further comprises at least one D-amino acid.
12. The composition of claim 11, further comprising a pharmaceutically
acceptable carrier, excipient, or diluent.
13. The composition of claim 12, wherein the majority of amino
acids comprising said compound are D-amino acids.
14. The composition of claim 13, wherein said membrane permeant
peptide is comprised entirely of D-amino acids.
15. A kit, comprising a compound comprising: a cell membrane-permeant
peptide consisting of a peptide sequence selected from the group
consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID
NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36; a metal chelation ligand;
and a non-functional linker moiety linking said peptide and said
metal chelation ligand; wherein said compound further comprises
at least one D-amino acid; and a reducing agent capable of reducing
a metal that can be coordinately incorporated into said metal chelation
ligand.
16. A method for imaging cells in vivo, comprising: administering
to an animal a cell imaging effective amount of a compound comprising:
a cell membrane-permeant peptide consisting of a peptide sequence
selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7,
SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36;
a chelated radionuclide or a chelated relaxivity metal; and non-functional
linker moiety linking said peptide and said chelated radionuclide
or said chelated relaxivity metal, wherein said compound further
comprises at least one D-amino acid; and monitoring the location
of said radionuclide or relaxivity metal within said animal.
17. A method for detecting cellular apoptosis in viva comprising:
administering to an animal a cellular apoptosis detecting effective
amount of a compound comprising: a cell membrane-permeant peptide
consisting of a peptide sequence selected from the group consisting
of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 34, SEQ
ID NO: 35, and SEQ ID NO: 36; a diagnostic substances; a non-functional
linker moiety linking the peptide and the diagnostic substance,
wherein said compound further comprises at least one D-amino acid
and said diagnostic substance is reactive with caspase; and monitoring
the diagnostic substance within the animal.
18. A method for detecting an enzyme in a cell comprising: contacting
the cell with an enzyme detecting effective amount of a compound
comprising: a cell-permeant peptide consisting of a peptide sequence
selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7,
SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36;
a diagnostic substance; a non-functional linker moiety linking the
peptide and the diagnostic substance, wherein said compound further
comprises at least one D-amino acid and said diagnostic substance
is reactive with the enzyme; removing unreacted compound from the
locus of the cell so that the signal to noise ratio is sufficient
for diagnostic purposes; and monitoring the presence of the diagnostic
substance in the cell.
19. A method for delivering a pharmaceutically active substance
to a cell comprising, contacting the cell with an effective amount
of a compound comprising: a cell-membrane permeant peptide consisting
of a peptide sequence selected from the group consisting of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:
35, and SEQ ID NO: 36; a pharmaceutically active substance; and
a non-functional linker moiety linking the peptide and the pharmaceutically
active substance; wherein said compound further comprises at least
one D-amino acid.
20. A method for assessing the effect of a drug in altering the
expression or activity of an enzyme in a target cell, comprising:
contacting the target cell with a diagnostically effective amount
of a compound comprising: a cell membrane-permeant peptide consisting
of a peptide sequence selected from the group consisting of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:
35, and SEQ ID NO: 36; a diagnostic substance; a non-functional
linker moiety linking the peptide and the diagnostic substance;
wherein the compound further comprises at least one D-amino acid
clearing unreacted compound from the locus of the cell so that the
signal to noise ratio is sufficient for diagnostic purposes; and
monitoring or evaluating the diagnostic substance in the target
cell.
21. The compound of claim 1, wherein said cell membrane-permeant
peptide comprises amino acids selected from the group consisting
of proteogenic amino acids and non-proteogenic amino acids.
22. The compound of claim 1 wherein said cell membrane-permeant
peptide is SEQ ID No: 7.
23. The compound of claim 6, wherein said radionuclide is a radioactive
isotope of a metal selected from the group consisting of Tc-m99
, In-111, Ga-67, Ga-68, Cu-64, Ru-97, Cr-51, Co-57, Re-188, and
Re-186.
24. The composition of claim 11, wherein said composition comprises
from about 1 .mu.Ci to about 100 mCi of said compound when said
compound is conjugated to a radionuclide.
25. The composition of claim 12, wherein said composition comprises
about from about 1 mCi to about 50 mCi when said compound is conjugated
to a radionuclide.
26. The compound of claim 4, wherein said membrane-permeant peptide
comprises at least one D-amino acid.
27. The compound of claim 4, wherein the majority of the amino
acids comprising said membrane-permeant peptide are D-amino acids.
28. The compound of claim 4, wherein said membrane-permeant peptide
is comprised of D-amino acids.
29. A method for imaging cells in vivo, comprising: administering
to an animal a cell imaging effective amount of a compound comprising:
a cell membrane-permeant peptide consisting of a peptide sequence
selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7,
SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36;
a chelated radionuclide or a chelated relaxivity metal; and a functional
linker moiety linking said peptide and said chelated radionuclide
or said chelated relaxivity metal, wherein said functional linker
confers target cell specificity to said compound, and monitoring
the location of said radionuclide or relaxivity metal within said
animal.
30. A method for detecting cellular apoptosis in vivo comprising:
administering to an animal a cellular apoptosis detecting effective
amount of a compound comprising: a cell membrane-permeant peptide
consisting of a peptide sequence selected from the group consisting
of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 34, SEQ
ID NO: 35, and SEQ ID NO: 36; a diagnostic substances; a functional
linker moiety linking the peptide and the diagnostic substance,
wherein the functional linker moiety comprises a caspase-reactive
sequence; and monitoring the diagnostic substance within the animal.
31. A method for detecting an enzyme in a cell comprising: contacting
the cell with an enzyme detecting effective amount of a compound
comprising: a cell-permeant peptide consisting of a peptide sequence
selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7,
SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36;
a diagnostic substance; a functional linker moiety linking the peptide
and the diagnostic substance, wherein the functional linker moiety
comprises a sequence reactive with the enzyme; removing unreacted
compound form the locus of the cell so that the signal to noise
ratio is sufficient for diagnostic purposes; and monitoring the
presence of the diagnostic substance in the cell.
32. A method for delivering a pharmaceutically active substance
to a cell comprising, contacting the cell with an effective amount
of a compound comprising: a cell-membrane permeant peptide consisting
of a peptide sequence selected from the group consisting of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:
35, and SEQ ID NO: 36; a pharmaceutically active substance; and
a functional linker moiety linking the peptide and the pharmaceutically
active substance; wherein said compound further comprises at least
one D-amino acid and said functional linker moiety confers target
cell specificity to the compound.
33. A method for assessing the effect of a drug in altering the
expression or activity of an enzyme in a target cell, comprising:
contacting the target cell with a diagnostically effective amount
of a compound comprising: a cell membrane-permeant peptide consisting
of a peptide sequence selected from the group consisting of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:
35, and SEQ ID NO: 36; a diagnostic substance; a functional linker
moiety linking the peptide and the diagnostic substance; wherein
the functional linker moiety confers target cell specificity to
the compound, and which comprises a sequence capable of interacting
with the enzyme so as to release the diagnostic substance from the
compound into the interior of the cell; clearing unreacted compound
from the locus of the cell so that the signal to noise ratio is
sufficient for diagnostic purposes; and monitoring or evaluating
the diagnostic substance in the target cell.
34. A compound, comprising: a cell membrane-permeant peptide consisting
of a peptide sequence selected from the group consisting of SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:
35, and SEQ ID NO: 36; a diagnostic or pharmaceutically active substance;
and a functional linker moiety linking said peptide and said diagnostic
or pharmaceutically active substance, wherein said functional linker
moiety confers target cell specificity to said compound and said
permeant peptide comprises at least one positively charged amino
acid, or a pharmaceutically acceptable salt of said compound.
35. The compound of claim 34 wherein said permeant peptide comprises
a majority of positively charged amino acids.
36. The compound of claim 35 wherein said permeant peptide is comprised
entirely of positively charged amino acids.
37. The compound of claim 36 wherein said permeant peptide comprises
a majority of D-amino acids.
38. The compound of claim 1 wherein the sequence of said peptide
comprises at least one modification to increase the cationic charge
of the permeant peptide.
39. The compound of claim 1, wherein said cell membrane-permeant
peptide is selected from the group consisting of the permeant peptide
of SEQ ID NO: 33, the permeant peptide of SEQ ID NO: 34, the permeant
peptide of SEQ ID NO: 35, and the permeant peptide of SEQ ID NO:
36.
40. The peptide conjugate of claim 39, wherein said peptide conjugate
comprises at least one D-amino acid.
41. The compound of claim 39, wherein the majority of the amino
acids comprising said membrane-permeant peptide are D-amino acids.
42. The compound of claim 39, wherein said membrane-permeant peptide
is comprised of D-amino acids.
43. A compound, comprising: a cell membrane-permeant peptide; a
diagnostic or pharmaceutically active substance; and a functional
linker moiety linking said peptide and said diagnostic or pharmaceutically
active substance, wherein said functional linker moiety confers
target cell specificity to said compound and said permeant peptide
consists of a peptide sequence selected from the group consisting
of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36,
or a pharmaceutically acceptable salt of said compound.
Medical Patent Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention broadly relates to the field of medicine.
More specifically, the present invention relates to the fields of
medical imaging, diagnostics, and pharmaceutical therapy. The present
invention provides methods and compositions for medical imaging,
evaluating intracellular processes, radiotherapy of intracellular
targets, and drug delivery by the use of novel cell membrane-permeant
peptide conjugate coordination and covalent complexes having target
cell specificity. The present invention also provides kits for conjugating
radionuclides and other metals to the peptide coordination complexes.
2. Description of Related Art
Radiopharmaceuticals in Diagnosis and Therapy
Radiopharmaceuticals provide vital information that aids in the
diagnosis and therapy of a variety of medical diseases (Hom and
Katzenellenbogen, Nucl. Med. Biol. 24:485-498, 1997). Data on tissue
shape, function, and localization within the body are relayed by
use of one of the various radionuclides, which can be either free
chemical species, such as the gas .sup.133Xe or the ions .sup.123I.sup.-,
and .sup.201Tl.sup.-, covalently or coordinately bound as part of
a larger organic or inorganic moiety, the images being generated
by the distribution of radioactive decay of the nuclide. Radionuclides
that are most useful for medical imaging include .sup.11C (t.sub.1/2
20.3 min), .sup.13N (t.sub.1/2 9.97 min), .sup.15O (t.sub.1/2 2.03
min), .sup.18F (t.sub.1/2 109.7 min), .sup.64Cu (t.sub.1/2 12 h),
.sup.68Ga (t.sub.1/2 68 min) for position emission tomography (PET)
and .sup.67Ga (t.sub.1/2 68 min), .sup.99mTc (t.sub.1/2 6 h), .sup.123I
(t.sub.1/2 13 h) and .sup.201Tl (t.sub.1/2 73.5 h) for single photon
emission computed tomography (SPECT) (Hom and Katzenellenbogen,
Nucl. Med. Biol. 24:485-498, 1997).
SPECT and PET imaging provide accurate data on radionuclide distribution
at the desired target tissue by detection of the gamma photons that
result from radionuclide decay. The high degree of spatial resolution
of modem commercial SPECT and PET scanners enables images to be
generated that map the radionuclide decay events into an image that
reflects the distribution of the agent in the body. These images
thus contain anatomic and functional information useful in medical
diagnosis. Similarly, if the radionuclides decay in such a manner
as to deposit radiation energy in or near the target cells or tissues,
the same approach would enable therapeutically relevant doses of
radioactivity to be deposited within the tissues.
Many radiopharmaceuticals have been prepared whose tissue localizing
characteristics depend on their overall size, charge, or physical
state (Hom and Katzenellenbogen, Nucl. Med. Biol. 24:485-498, 1997).
Other radiopharmaceuticals are synthesized with the intention to
be ligands for specific hormone, neurotransmitter, cell surface
or drug receptors, as well as specific high affinity transport systems
or enzymes. As these receptors and enzymes are known to be involved
in the regulation of a wide variety of vital bodily functions, effective
imaging agents can be used in the diagnosis or staging of a variety
of disease states, in which such receptors are functioning abnormally
or are distributed in an abnormal fashion, or in the monitoring
of therapy (Hom and Katzenellenbogen, Nucl. Med. Biol. 24:485-498,
1997). Effective therapeutic agents can also be used to deliver
pharmacologically active doses of compounds to the same receptors
and enzymes.
Recent advances in molecular, structural and computational biology
have begun to provide insights in the structure of receptors and
enzymes that should be considered in the design of various ligands.
Two key issues derived from the structure and distribution of these
receptors have a direct impact on the development of new radiopharmaceuticals:
1) the location of a receptor or enzyme activity in the body (i.e.,
peripheral sites versus brain sites), and 2) its subcellular location
(i.e., on the cell surface versus intracellular) will determine
whether a radiopharmaceutical injected intravenously will need to
traverse zero, one, two or more membrane barriers to reach the target.
The structure of the receptor and the nature of its interaction
with the ligand will determine the degree to which large ligands
or ligands with large substituents may be tolerated (Hom and Katzenellenbogen,
Nucl. Med. Biol. 24:485-498, 1997). For example, radiopharmaceuticals
which target cell surface receptors will encounter no membrane barriers
to reach their target. Natural ligands for these receptors can be
large, and often are charged and, consequently, large radiopharmaceuticals
are tolerated. Conversely, for a radiopharmaceutical to reach intracellular
receptors or enzymes, at least one membrane barrier, the cell plasma
membrane, must be traversed, and if the target site is within the
central nervous system, the radiopharmaceutical must also traverse
the plasma membranes of endothelial cells of the brain which constitute
the blood-brain barrier. Such a situation usually favors radiopharmaceutical
designs that strongly minimize ligand size and molecular weight
(Hom and Katzenellenbogen, Nucl. Med. Biol. 24:485-498, 1997). Thus,
as the number of membrane barriers increases, a premium is placed
on keeping the size of a conventional radiopharmaceutical small
(<600 Da) and the lipophilicity intermediate (characterized by
an octanol-water partition coefficient, log P .about.2) to enable
the agent to traverse membranes (Dishino, et al., J Nucl Med 24:
1030-1038, 1983; Papadopoulos, et al., Nucl Med Biol 20:101-104,
1993; Eckelman, Eur J Nucl Med 22:249-263, 1995). This has conventionally
precluded the use of peptide radiopharmaceuticals for intracellular
targets.
There has been a great deal of research into the development of
radiopharmaceuticals directed toward cell surface receptors whose
natural ligands are peptides. Tc-labeled peptides can span the spectrum
of size. The derivatizing group or chelation core of smaller peptides
has been reported to impact the in vitro binding and in vivo distribution
properties of these compounds (Babich and Fischman, Nucl Med Biol
22:25-30, 1995; Liu, et al., Bioconj Chem 7:196-202, 1996). For
larger peptides or proteins, the labeling process can usually occur
at one or more of several reactive sites, and thus, the final mixture
of compounds is less chemically defined. Thus, for larger proteins,
it is usually much less clear which of these sites, if any, might
be more favorable for receptor interaction and whether or not specific
labeling would increase biological activity of the agent (Hom and
Katzenellenbogen, Nucl. Med. Biol. 24:485-498, 1997).
It is known that low molecular weight peptides and antibody fragments
provide rapid tumor targeting and uniform distribution in tumor
tissues (Yokota et al., Cancer Res 53:3776-3783, 1993). While such
characteristics render low molecular weight peptides attractive
vehicles for the delivery of radioactivity to tumor tissues and
organs for both targeted imaging and radiotherapy, nonetheless problems
have been encountered. High and persistent localization of the radioactivity
is observed in the kidneys, which compromises tumor visualization
in the kidney region and limits therapeutic potential (Buijs, et
al., J Nucl Med 33:1113-1120, 1992; Baum, et al., Cancer (Phila)
73:896-899, 1994; Choi, et al., Cancer Res 55:5323-5329, 1995; Behr,
et al., J Nucl Med 36:430-441, 1995). As discussed by Arano, et
al. (Cancer Res 59:128-143, 1999), radiolabeled low molecular weight
peptides and antibody fragments would become much more useful for
targeted imaging and therapy if the renal radioactivity levels could
be reduced without impairing those in the target tissue. Previous
studies have indicated that radiolabeled low molecular weight peptides
and antibody fragments are likely resorbed by proximal tubules via
luminal endocytosis after glomerular filtration (Silberbagl, S.
Physiol Rev 68:811-1007, 1988). The long residence times of the
radiometabolites generated after lysosomal proteolysis of the radio
labeled fragments in renal cells were also reported to be responsible
for the persistent renal radioactivity levels (Choi, et al., Cancer
Res 55:5323-5329; Rogers, et al., Bioconjugate Chem 7:511-522,1996).
There exists a continued need for peptide-based radiopharmaceuticals
that are rapidly cleared and target intracellular receptors or enzyme
activities.
Peptide-Based Metal Coordination Complexes
Small peptides can be readily prepared by automated solid phase
peptide synthesis (Merifield et al., Biochemistry 21:5020-5031,
1982; Houghten, Proc Natl Acad Sci USA 82:5131-5135, 1985; Lin,
et al., Biochemistry 27:5640-5645, 1988) using anyone of a number
of well known, commercially available automated synthesizers, such
as Applied Biosystems ABI 433A peptide synthesizer. Many combinations
of natural and non-natural amino acids and peptide sequence mimetics
(peptidomimetics) are possible, and selective engineering of favorable
target-binding and pharmacokinetic properties can be accomplished
with natural and unnatural peptides (Lister-James et al., Q. J:
Nucl. Med., 41:111-118, 1997). Peptidomimetics are unnatural biopolymers
that do not contain .alpha.-amino acids, but rather incorporate
backbone structures with hydrogen-bonding groups (such as urea),
chiral centers, side chain functionalities, and a sufficient degree
of conformational restriction to behave similar to, or mimic the
bioactivities of, a natural polypeptide. Peptide-based imaging agents
are also well known (Lister-James et al., Q. J: Nucl. Med., 41:111-118,1997;
Lister-James et al., J. Nucl. Med., 38:105-111, 1997), especially
those that incorporate Tc-99m as the radionuclide, the most commonly
used isotope in medical imaging.
The metallic character of Tc-99m requires that it be stabilized
by a chelation system to be coupled to an imaging agent. This chelator
may typically involve a multiple heteroatom coordination system,
or the formation of a non-labile organometallic species. There are
two broad strategies for binding metals for biological applications.
These are "the pendant approach" and "the integrated
approach," which have been recently reviewed by Katzenellenbogen
and colleagues (Hom and Katzenellenbogen, Nucl. Med. Biol., 24:485-498,
1997). The pendant (or conjugate) approach involves the strategic
placement of a Tc-99m-chelator-tether moiety at a site on the ligand
that will not hinder binding of the ligand to its high affinity
receptor. The integrated approach replaces a component of a known
high-affinity receptor ligand with the requisite Tc-99m chelator
such that there is a minimal change in the size, shape, structure,
and binding affinity of the resultant molecule. Applications involving
peptide-based imaging agents typically use the conjugate design,
whereby an appropriate metal chelating moiety is affixed to the
amino or carboxy terminus of the targeting peptide.
A variety of metal chelation systems have been developed for synthesis
of radioisotopic and magnetic resonance peptide-based imaging agents.
Peptide-based agents target extracellular or externally oriented
membrane bound receptors (Hom and Katzenellenbogen, Nucl. Med. Biol.,
24:485-498, 1997) because the charge, size, and pharmacokinetic
properties of typical peptide structures do not allow diffusion
across the lipid bilayer of the cell plasma membrane. This limitation
has prevented peptide metal chelates from reporting the functional
status or biological activity of intracellular receptors or enzymes
or other homeostatic activities and intracellular targets. Although
techniques and reagents for labeling antibodies and antibody fragments
with metal-chelates are well known in the art (Hom and Katzenellenbogen,
Nucl. Med. Biol., 24:485-498, 1997, and references therein), they
target extracellular or externally oriented cell surface receptors.
Tat Proteins and Peptides
Tat is an 86-amino acid protein involved in the replication of
human immunodeficiency virus type 1 (HIV-1). The HIV-1 Tat transactivation
protein is efficiently taken up by cells (Mann and Frankel, EMBO,
10:1733-1739, 1991; Vives et al., J. Virol., 68:3343-3353, 1994),
and low concentrations (nM) are sufficient to transactivate a reporter
gene expressed from the HIV-1 promoter (Mann and Frankel, EMBO,
10:1733-1739, 1991). Exogenous Tat protein is able to translocate
through the plasma membrane and reach the nucleus to transactivate
the viral genome (Frankel and Pabo, Cell 55:1189-1193, 1988; Ruben,
et al, J Virol 63:1-8, 1989; Garcia, et al., EMBO J 7:3143, 1988;
Jones, Genes Dev 11:2593-2599,1997).
A region of the Tat protein centered on a cluster of basic amino
acids is responsible for this translocation activity (Vives et al.,
J Biol. Chem., 272:16010-16017, 1997). Tat peptide-mediated cellular
uptake and nuclear translocation have been demonstrated in several
systems (Vives, et al., J Biol Chem 272:16010-16017, 1997; Jones,
Genes Dev 11:2593-2599, 1997). Chemically coupling a Tat-derived
peptide (residues 37-72) to several proteins results in their internalization
in several cell lines or tissues (Fawell, et at, Proc Natl Acad
Sci USA 91:664-668, 1994; Anderson, et at, Biochem Biophys Res Commun
194:876-8884, 1993; Fahraeus, et al., Curr Biol 6:84-91, 1996; Nagahara,
et al, Nat Med 4:1449-1452, 1998). A synthetic peptide consisting
of the Tat basic amino acids 48-60 with a cysteine residue at the
C-terminus coupled to fluorescein maleimide translocates to the
cell nucleus as determined by fluorescence microscopy (Vives et
al., J. Biol. Chem., 272:16010-16017, 1997). In addition, a fusion
protein (Tat-NLS-.beta.-Gal) consisting of Tat amino acids 48-59
fused by their amino-terminus to .beta.-galactosidase amino acids
9-1023 translocates to the cell nucleus in an ATP-dependent, cytosolic
factor-independent manner (Efthymiadis et al., J. Biol. Chem., 273:1623-1628,
1998).
While the literature teaches that Tat peptide constructs and similar
membrane permeant peptides readily translocate into the cytosolic
and nuclear compartments of living cells, little is known regarding
the cellular retention characteristics over time once the permeant
peptide constructs are no longer in contact with the cell surface
from the extracellular fluid spaces. Furthermore, no information
is available regarding the pharmacokinetic and distribution characteristics
of membrane-permeant peptides within a whole living organism, animal
or human.
Apoptosis
Chemotherapeutic drugs used in the treatment of cancer are thought
to interact with diverse cellular targets in conferring lethal effects
on mammalian cells. Recently, anticancer agents, irrespective of
their intracellular target, have been shown to exert their biological
effect in target cells by triggering a common final death pathway
known as apoptosis (Fulda, et al., Cancer Res 57:3823-3829,1997;
Fisher, Cell 78:539-542, 1994). Thus, there exists mounting evidence
that many anticancer treatments may kill through apoptosis by activating
intracellular death machinery in the target cell rather than by
simply crippling various components of cellular metabolism (Fulda,
et al., Cancer Res 57:3823-3829,1997; Fisher, Cell 78:539-542, 1994).
In fact, the action of ionizing radiation, drug therapy, and withdrawal
of physiological survival factors all appear to act as death stimuli
in promoting execution of this common apoptotic pathway (Evan and
Littlewood, Science 281:1317-1322, 1998; Ashkenazi and Dixit, Science
281:1305-1308, 1998). Thus, new models of resistance to therapy
have begun to focus on mechanisms that antagonize execution of the
apoptotic pathway.
Apoptotic stimuli can arise from the nucleus, cell membrane surface,
or the mitochondria (Wyllie, Nature, 389:237-38, 1997). Ultimately,
the stimuli converge on a process of activation of a family of interleukin
1.beta.-converting enzymes {(ICE)-like cysteine proteases} known
as cysteine aspartases ("caspases") (Thornberry et al.,
Science, 281:1312-16, 1998). Members of the caspase family are activated
in apoptosis and have been shown to be necessary for programmed
cell death in a number of biological systems (Yuan et al., Cell,
75:641-52, 1993; Thornberry et al., Science, 281:1312-16,1998).
The caspase gene family, defined by sequence homology, is also characterized
by conservation of key catalytic and substrate-recognition amino
acids (Talanian et al., J. Biol. Chem., 272:9677-82, 1997). Thirteen
mammalian caspases (1 through 13) have thus far been isolated, having
distinct roles in apoptosis and inflammation (Thornberry et al.,
Science, 281:1312-16, 1998). In apoptosis, some caspases are involved
in upstream regulatory events and are known as "initiators,"
while others are directly responsible for proteolytic cleavages
that lead to cell disassembly and are known as "effectors."
Evidence indicates that caspases transduce or amplify signals by
mutual activation. For example, Fas-induced apoptosis is characterized
by an early, transient caspase-1-like protease activity followed
by a caspase-3-like activity, suggesting an ordered activation cascade
(Enari et al., Nature, 380:723-26, 1996). Other data suggest that
both caspase-3 and caspase-7 are activated by caspase-6 and caspase-10
(Thornberry et al., Science, 281:1312-16, 199; Fernandes-Alnemri,
Proc. Natl. Acad. Sci. USA, 93:7464-69, 1996). Thus, while the activation
cascade hypothesis remains to be absolutely proven (Villa et al.,
Trends in Biochem. Sci., 22:388-93, 1997), circumstantial evidence
strongly points to caspase-3 as one key "effector" caspase,
standing at the center of the execution pathway of the cell death
program.
Caspases are some of the most specific of the proteases, showing
an absolute requirement for cleavage after aspartic acid (Thornberry
et al., Science, 281:1312-16, 1998). Recognition of at least four
amino acids, amino terminal to the cleavage site, is also necessary
for efficient catalysis. The preferred recognition motif differs
significantly between caspases, thereby contributing to their biologically
diverse functions (Talanina et al., J. Biol. Chem. 272:9677-82,
1997). In addition to high specificity, caspases are also highly
efficient, with K.sub.cat/K.sub.m values >10.sup.6 M.sup.-1s.sup.-1
(Thornberry et al., Science, 281:1312-16, 1998). When viewed from
the perspective of a molecular target for oncological imaging, this
is a key property of the caspases that allows detection of caspase
activity in vivo by radiosubstrates. Another advantage of the caspases
as imaging targets centers on the nature of the biochemical reaction.
Because normal cells have essentially non-detectable levels of caspase
activity, and once activated, the "caspase cascade" amplifies
reaction rates to maximal velocities (Thornberry et al., Science,
281:1312-16, 1998), the signal readout obtained by imaging is binary
in character. That is, in the absence of caspase activity, the imaging
signal will be low, and when activated, a highly amplified imaging
signal will result. This renders the caspase-mediated enzymatic
reaction essentially zero-order in situ and, therefore, independent
of radiotracer concentration or specific activity, thus eliminating
the complexities of first or higher order reaction rates.
Deregulation of apoptosis resulting in insufficient cell death
can occur in cancer, allowing malignant tissues to grow (Thornberry
et al., Science, 281:1312-16, 1998). Conversely, some diseases involve
excess apoptosis, such as neurodegenerative disease, ischemia-reperfusion,
graft-vs-host disease, and autoimmune disorders (Thornberry et al.,
Science, 281: 1312-16, 1998). Accordingly, two-fold strategies for
therapeutic intervention are actively underway within the pharmaceutical
industry, one to selectively induce apoptosis through caspase activation,
the other to inhibit caspase activity. In order to assess the treatments
to alter apoptosis, an accurate means to assess apoptoic activity
in vivo is needed.
Inactive pro-caspases are constitutively expressed as pro-enzymes
in nearly all cells, existing in latent forms in the cell cytoplasm
(Villa et al., Trends in Biochem. Sci. 22:388-93, 1997). Thus, while
caspase-3 can be readily identified by Western blots, this requires
biopsy material and lysis of the cells. Furthermore, activation
of caspase-3 is only inferred by observation of lower molecular
weight cleavage fragments on the blot. Activation of caspase-3 has
also been inferred from nuclear shifts of antigen by immunohistochemical
analysis of biopsy material and shown to be associated with a more
favorable prognosis in, for example, pediatric neuroblastoma (Nakagawara
et al., Cancer Res. 57:4578-84, 1997). However, these indirect methods
only imply activation. Thus, the simple determination of the presence
or absence of caspase proteins is not necessarily diagnostically
useful. A method to directly and non-invasively detect and quantify
the enzymatic activity of caspases in order to monitor the commitment
to cell death pathway is needed. Because caspases are cytosolic
enzymes, new diagnostic and therapeutic compounds are required that
can readily cross cell membranes, and whose specificity is based
on the presence of protease activity.
Tat Peptide Complexes
Frankel et al. (U.S. Pat. Nos. 5,804,604; 5,747,641; 5,674,980;
5,670,617; 5,652,122) discloses the use of Tat peptides to transport
covalently linked biologically active cargo molecules into the cytoplasm
and nuclei of cells. Frankel only discloses covalently linked cargo
moieties, and does not teach or suggest the attachment of metals
to Tat peptides by metal coordination complexes. Specifically, Frankel
does not teach the use of peptide chelators to introduce radioimaging
materials into cells. In addition, while Frankel teaches the use
of cleavable coupling reagents between the Tat protein and the cargo
molecule, the cleavable linkers disclosed are non-specific, such
that the retention of the cargo molecule is not limited to specific
cells.
Anderson et al. (U.S. Pat. Nos. 5,135,736 and 5,169,933) discloses
the use of covalently linked complexes (CLCs) to introduce molecules
into cells. CLCs comprise a targeting protein, preferably an antibody,
a cytotoxic agent, and an enhancing moiety. Specificity is imparted
to the CLC by means of the targeting protein, which binds to the
surface of the target cell. After binding, the CLC is taken into
the cell by endocytosis and released from the endosome into the
cytoplasm. In one embodiment, Anderson discloses the use of the
Tat protein as part of the enhancing moiety to promote translocation
of the CLC from the endosome to the cytoplasm. In another embodiment,
Anderson discloses the use of CLCs to transport radionuclides useful
for imaging into cells. The complexes described by Anderson are
limited in their specificity to cells that can be identified by
cell surface markers. Many biologically and medically significant
cellular processes, for example caspase protease activities discussed
above, are not detectable with cell surface markers. In addition,
the attachment of enhancing moieties to the CLC is accomplished
by the use of bifunctional linkers. The use of bifunctional linkers
results in the production of a heterogeneous population of CLCs
with varying numbers of enhancing moieties attached at varying locations.
This can lead to the production of CLCs in which the biological
activity of the targeting protein, the enhancing moiety, or both,
are lost. Another disadvantage of CLCs is that the number and location
of linked enhancing moieties will vary with each reaction, so that
a consistent product is not produced.
There is a need in the art for cell membrane-permeant peptide complexes
of uniform composition, capable of delivering radionuclides, other
metals, diagnostic substances such as fluorochromes, dyes, etc.,
and therapeutic and cytotoxic drugs into cells in a specific and
selective manner. Furthermore, rapid clearance of the complexes
from non-target cells and tissues of the body would facilitate and
enhance the utility of such complexes in vivo.
SUMMARY OF THE INVENTION
The present inventor has surprisingly discovered that the addition
of D-amino acid containing membrane-permeant peptides attached to
non- or poorly permeant drugs, diagnostic and/or therapeutic substances
such as oligonucleotides, peptides, peptide nucleic acids, fluorochromes,
dyes, enzyme substrates, and metals useful in medical therapy, imaging,
and/or diagnostics greatly increases their accumulation within cells.
As shown in Example 14, this increase in accumulation is on the
order of 8- to 9-fold as compared to membrane-permeant peptides
comprising only naturally occurring L-amino acids. Thus, use of
the D-amino acid containing membrane-permeant peptides of the present
invention allows delivery of greater amounts of therapeutic or diagnostic
substances to the interior of cells either in vivo or in vitro than
was heretofore possible using membrane permeant peptides containing
only L-amino acids.
The present inventor has also discovered that the Tat peptide and
other cell membrane-permeant peptides can be used to selectively
deliver non- or poorly permeant drugs, diagnostic and/or therapeutic
substances such as oligonucleotides, peptides, peptide nucleic acids,
fluorochromes, dyes, enzyme substrates, and metals useful in medical
therapy, imaging, and/or diagnostics selectively to cells in vivo
only when functional linkers are introduced into the permeant peptide
construct, and has developed methods for linking these substances
to Tat and other peptides for use in such methods. As illustrated
in Examples 6 and 10, below, non-targeted Tat peptides, rather than
being trapped inside cells and tissues indefinitely, are cleared
surprisingly rapidly from body tissues when introduced into the
living organism. Furthermore, non-functionalized prototypes of such
complexes are rapidly excreted by the kidneys and cleared from the
whole body. Thus, membrane-permeant peptides covalently linked to
oligopeptides, proteins, oligonucleotides, and drugs as known previously
possess rapid and ineffective biological half-times within the whole
organism.
Thus, in response to this surprising and unanticipated property
of D-amino acid containing permeant peptides and to improve upon
the prior art, the present invention provides novel permeant peptide
conjugates, complexes and methods that possess the advantage of
enabling the targeted trapping of greater amounts of such compounds
or fragments thereof within desired cells, tissues and organs of
the intact body of living organisms. Conversely, when it is desired
to increase the rates of clearance of cargo oligopeptides, proteins,
oligonucleotides, metals, and drugs, the present invention also
provides methods that will enhance their rates of clearance from
the body.
Accordingly, in a first aspect, the present invention provides
a compound comprising a cell membrane-permeant peptide; a diagnostic
or pharmaceutically active substance; and a functional linker moiety
linking the peptide and the diagnostic or pharmaceutically active
substance, wherein the compound further comprises at least one D-amino
acid and the functional linker moiety confers target cell specificity
to the compound, or a pharmaceutically acceptable salt of the compound.
In a second aspect, the present invention provides a composition
comprising, a compound comprising a cell membrane-permeant peptide;
a diagnostic or pharmaceutically active substance; and a functional
linker moiety linking the peptide and the diagnostic or pharmaceutically
active substance, wherein the compound further comprises at least
one D-amino acid and the functional linker moiety confers target
cell specificity to the compound. The composition can further comprise
a pharmaceutically acceptable carrier, excipient, or diluent.
In a third aspect, the present invention provides a kit comprising,
a compound comprising a cell membrane-permeant peptide; a metal
chelation ligand; and a functional linker moiety linking the peptide
and the metal chelation ligand, wherein the compound further comprises
at least one D-amino acid and the functional linker moiety confers
target cell specificity to the compound, and a reducing agent capable
of reducing a metal that can be coordinately incorporated into the
metal chelation ligand.
In another aspect, the present invention provides a method for
imaging cells in vivo, comprising administering to an animal a cell
imaging effective amount of a compound comprising a cell membrane-permeant
peptide; a chelated radionuclide or a chelated relaxivity metal;
and a functional linker moiety linking the peptide and the chelated
radionuclide or the chelated relaxivity metal, the functional linker
confering target cell specificity to the compound, and monitoring
or evaluating the location of the radionuclide or relaxivity metal
within the animal. Further the compound may comprise at least one
D-amino acid.
In another aspect, the present invention provides a method for
imaging cells in vitro, comprising contacting the cells with a cell
imaging effective amount of a compound comprising a cell membrane-permeant
peptide; a diagnostic substance; and a functional linker moiety
linking the peptide and the diagnostic substance, wherein the functional
linker confers target cell specificity to the compound, and monitoring
or evaluating the presence of the diagnostic substance within the
cells. Further, the compound may comprise at least one D-amino acid.
In a further aspect, the present invention provides a method for
detecting cellular apoptosis in vivo, comprising administering to
an animal a cellular apoptosis detecting effective amount of a compound
comprising a cell membrane-permeant peptide; a diagnostic substance;
and a functional linker moiety linking the peptide and the diagnostic
substance, wherein the functional linker moiety comprises a caspase-reactive
sequence, and monitoring the diagnostic substance within the animal.
Further, the compound may contain at least one D-amino acid.
In another aspect, the present invention provides a method for
detecting cellular apoptosis in vitro, comprising contacting cells
or tissue in vitro with a cellular apoptosis detecting effective
amount of a compound comprising a cell membrane-permeant peptide;
a diagnostic substance; and a functional linker moiety linking the
peptide and the diagnostic substance, wherein the functional linker
moiety comprises a caspase-reactive sequence, and monitoring the
diagnostic substance within the cells or tissue. Further, the compound
may contain at least one D-amino acid.
In yet another aspect, the present invention provides a method
for detecting an enzyme in a cell, comprising contacting the cell
with an enzyme detecting effective amount of a compound comprising
a cell membrane-permeant peptide; a diagnostic substance; a functional
linker moiety linking the peptide and the diagnostic substance,
wherein the functional linker moiety comprises a sequence reactive
with the enzyme; removing unreacted compound from the locus of the
cell so that the signal to noise ratio is sufficient for diagnostic
purposes; and monitoring the presence of the diagnostic substance
in the cell. Such monitoring can be performed quantitatively, and
the cell can be present within a living animal. Furthermore, the
enzyme can be one that is characteristically associated with a disease,
condition, or disorder. Further, the compound may contain at least
one D-amino acid.
In yet another aspect, the present invention provides a method
for diagnosing the presence of a disease, condition, or disorder
in an animal, comprising administering to the animal a diagnostically
effective amount of a compound comprising a cell membrane-permeant
peptide; a diagnostic substance; a functional linker moiety linking
the peptide and the diagnostic substance, wherein the functional
linker moiety confers target cell specificity to the compound, and
which comprises a sequence reactive with an enzyme indicative or
characteristic of the disease, condition, or disorder, and monitoring
the diagnostic substance within the animal. By way of example, the
disease, condition, or disorder can be a cancer such as a central
nervous system tumor, breast cancer, liver cancer, lung cancer,
head cancer, neck cancer, a lymphoma, or a melanoma. Further, the
compound may contain at least one D-amino acid.
In still another aspect, the present invention provides a method
of assessing the effectiveness of cancer therapy, comprising administering
to an animal undergoing cancer therapy a diagnostically effective
amount of a compound comprising a cell membrane-permeant peptide;
a diagnostic substance; and a functional linker moiety linking the
peptide and the diagnostic substance, wherein the functional linker
moiety confers target cell specificity to the compound, and which
comprises a caspase-reactive sequence, and monitoring the diagnostic
substance within the animal. Such monitoring can be performed quantitatively.
Furthermore, the method can be repeated at intervals during the
cancer therapy, and the quantity of the diagnostic substance detected
within the animal at each interval can be compared to the quantity
of the diagnostic substance detected at previous intervals to determine
the effectiveness of the therapy. In addition, the compound may
contain at least one D-amino acid.
In yet another aspect, the present invention provides a method
of delivering a pharmaceutically active substance to a cell, comprising
contacting the cell with an effective amount of a compound comprising
a cell membrane-permeant peptide; a pharmaceutically active substance;
and a functional linker moiety linking the peptide and the pharmaceutically
active substance, wherein the compound further comprises at least
one D-amino acid and the functional linker moiety confers target
cell specificity to the compound.
In another aspect, the present invention provides a method of treating,
inhibiting, or preventing a disease, condition, or disorder responsive
to treatment with a pharmaceutically active substance in an animal,
comprising administering to the animal a pharmaceutically effective
amount of a compound comprising a cell membrane-permeant peptide;
a pharmaceutically active substance; and a functional linker moiety
linking the peptide and the pharmaceutically active substance, wherein
the compound further comprises at least one D-amino acid and the
functional linker moiety confers target cell specificity to the
compound.
In another aspect, the present invention provides a method for
selectively destroying cells expressing a selected enzyme activity,
comprising contacting the cells with a cell-destroying effective
amount of a compound comprising a cell membrane-permeant peptide;
a cytotoxic substance; and a functional linker moiety linking the
peptide and the cytotoxic substance, wherein the compound further
comprises at least one D-amino acid and the functional linker moiety
confers target cell specificity to the compound.
In yet another aspect, the present invention provides a method
for assessing the effect of a drug in altering the expression or
activity of an enzyme in a target cell, comprising contacting the
target cell with a diagnostically effective amount of a compound
comprising a cell membrane-permeant peptide; a diagnostic substance;
a functional linker moiety linking the peptide and the diagnostic
substance, wherein the functional linker moiety confers target cell
specificity to the compound, and which comprises a sequence capable
of interacting with the enzyme so as to release the diagnostic substance
from the compound into the interior of the cell; clearing unreacted
compound from the locus of the cell so that the signal to noise
ratio is sufficient for diagnostic purposes; and monitoring or evaluating
the diagnostic substance in the target cell. Such monitoring can
be performed quantitatively, and the target cell can be present
within a living animal. Furthermore, the enzyme can be associated
with a disease, condition, or disorder. In addition, the compound
may further comprise at least one D-amino acid.
In yet another aspect, the present invention provides a method
for detecting the expression of a nucleic acid sequence, which can
be DNA or RNA, encoding an enzyme, a receptor, or a binding protein
introduced into a cell, comprising contacting the cell with a compound
comprising a cell membrane-permeant peptide; a diagnostic substance;
a functional linker moiety linking the peptide and the diagnostic
substance, wherein the functional linker moiety confers target cell
specificity to the compound, and which comprises a sequence capable
of interacting with the enzyme, receptor, or binding protein so
as to selectively retain the diagnostic substance in the cell, and
monitoring the diagnostic substance in the cell. Further, the compound
may comprise at least one D-amino acid.
Further scope of the applicability of the present invention will
become apparent from the detailed description and drawings provided
below. However, it should be understood that the following detailed
description and examples, while indicating preferred embodiments
of the invention, are given by way of illustration only since various
changes and modifications within the spirit and scope of the invention
will become apparent to those skilled in the art from this detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects, features, and advantages of the present
invention will be better understood from the following detailed
description taken in conjunction with the accompanying drawings,
all of which are given by way of illustration only, and are not
limitative of the present invention, in which:
FIG. 1 shows the general structure of a cell membrane-permeant
peptide coordination complex of the present invention.
FIG. 2 shows the proposed structure of an oxotechnetium-Tat-peptide
complex. The coordination metal (Tc.sup.vO) may be replaced by Re.sup.vO
to form essentially isostructural complexes.
FIG. 3 shows the time course of cellular uptake of a Tc-99m-Tat
peptide complex in human Jurkat cells. Extracellular concentration
of peptide was 950 nM. Each point represents the mean of 4 observations.+-.SEM
when larger than the symbol. Cell accumulation of the Tc-99m-Tat
peptide complex is 90% complete within 2 minutes and established
a quasi-steady state that was maintained for at least 1 hour (data
not shown).
FIG. 4 shows the concentration-dependence of plateau accumulation
of Tc-99m-Tat peptide conjugate into human Jurkat cells. Each point
represents the mean of 4 observations.+-.SEM when larger than the
symbol.
FIG. 5 shows washout kinetics of a non-functional Tc-99m-Tat peptide
complex from human Jurkat cells. Cells were loaded to plateau uptake
(.about.30 min), washed in ice cold buffer to clear extracellular
spaces, and then bathed in isotope-free buffer at 37.degree. C.
for the times indicated. Cell-associated counts are shown. Each
point represents the mean of 4 observations.+-.SEM when larger than
the symbol.
FIG. 6 shows the cellular accumulation of Tat peptide chelate conjugates
in KB-3-1 human tumor cells. KB-3-1 cells were incubated with compound
for 15 min at room temperature followed by a rapid wash and fixation:
fluorescein maleimide (0.5 .mu.M) alone (left) or Tat peptide chelate-fluorescein
maleimide conjugate (right). Tat peptide chelate was conjugated
with fluorescein maleimide on the C-terminal Cys residue. There
was no counter staining of nuclei with propidium iodide in this
example. Note the distribution of fluorescence from labeled peptide
conjugate corresponding to cytosolic and nuclear (nucleolar) distribution.
Bar=5 .mu.m.
FIG. 7 shows RP-HPLC traces (440 nm) of cell lysates from control
untreated Jurkat cells without added Tat peptide (A), untreated
Jurkat cells incubated in fluorescein tagged Tat peptide (B), and
ceramide-treated caspase-3 activated cells incubated in fluorescein
tagged Tat peptide (C). The intact fluorescein tagged Tat peptide
is seen in tracing B (arrow at R.sub.t=33.5 min). In tracing C,
note the absence of the intact Tat peptide. All three tracings show
autofluorescent compounds present in the cells at R.sub.t=22 and
28 min.
FIG. 8 shows scintigraphic image of rapid renal excretion of a
Tc-99m-Tat peptide in a normal FVB mouse 30 minutes post injection.
Following metofane anesthesia, Tc-99m-Tat chelate (200 .mu.Ci, prepared
as described in the application) was administered by tail vein injection
and the mouse immediately positioned for imaging on a gamma scintillation
camera (Siemens Basicam; 5 mm pinhole collimator; 20% energy window
centered over 140 keV). Sequential posterior images of the mouse
were collected at one frame/minute for .about.30 min with a 128.times.128
matrix. A [mal 5 minute acquisition with a 256.times.256 matrix
was also obtained. Images were corrected for radioactive decay,
but no corrections were made for scatter or attenuation. While radioactivity
initially distributed throughout the body, note focal radioactivity
within the urinary bladder after only 30 minutes, reflecting rapid
renal excretion of the Tat peptide conjugate.
FIG. 9 shows scintigraphic images of organ distribution of caspase-3-cleavable
Tc-99m-Tat peptide in FVB mice 30 minutes post injection. Using
a published procedure (Blankenberg, et al., Proc Natl Acad Sci USA
95:6349-6354, 1998), FVB mice were administered purified hamster
anti-Fas mAb (Jo2, PharMingen; 8 .mu.g/animal) by i.v. injection
and allowed to recover for 45 minutes prior to imaging. Following
metofane anesthesia, Tc-99m-Tat chelate (200 .mu.Ci, prepared as
described in the text) was administered by tail vein injection and
mice immediately positioned for imaging on a gamma scintillation
camera (Siemens Basicam; 5 mm pinhole collimator; 20% energy window
centered over 140 keV). Sequential posterior images of mice were
collected at one frame/minute for .about.30 min with a 128.times.128
matrix. A final 5 minute acquisition with a 256.times.256 matrix
was also obtained. Images were corrected for radioactive decay,
but no corrections were made for scatter or attenuation. Left, untreated
control mouse; right, mouse pre-treated with anti-Fag mAb. Note
focal radioactivity only in the urinary bladder of the control mouse,
but abundant retention of radioactivity in the pre-treated animal
within the liver and kidneys, two organs that express the Fas receptor
wherein caspase-mediated apoptosis is induced and imaged.
FIG. 10 shows comparative uptake of D, L and mixed D/L [.sup.99mTc]Tat-peptide
chelate conjugates. Net, 20 minute accumulation values into Jurkat
cells are shown. Each bar represents the mean of 4 observations+SEM.
(1) L/L, [.sup.99mTc]Tat-peptide conjugate 2; (2) L/D [.sup.99mTc]Tat-peptide
conjugate 5; (3) D/D [.sup.99mTc]Tat-peptide conjugate 9; and (4)
D/L [.sup.99mTc]Tat-peptide conjugate 12.
FIG. 11 shows net cell uptake of permeation peptides with varying
lengths of the permeation sequence. Radiolabeled peptides were incubated
with Jurkat cells as described in FIG. 1 and Methods. A=D Tat basic
domain (13-17), B=D amphipathic cationic peptide (18-21), C=L poly-Arg
peptide (26, 28, 30, 32), D=D poly-Arg peptide (27, 29, 31, 33);
(.box-solid.) 9 residues in permeation sequence, (.quadrature.)
8 residues, (vertical lines) 7 residues, (horizontal lines) 6 residues,
(checkered lines) 5 residues.
FIG. 12A shows the effect on Jurkat cell uptake of substituting
different amino acids for Gln in Tat basic domain (RKKRRXRRR); X=Glu,
24; Gln, 7; Asn, 22; Norleu, 25; and Orn, 23. FIG. 12B shows the
effect on Jurkat cell uptake of a single substitution in poly-Arg.sub.8
peptide (RRRRXRRR); X=Arg, 31; Orn, 35; Norleu, 37; Asn, 34; Glu,
36.
DETAILED DESCRIPTION OF THE INVENTION
The following detailed description is provided to aid those skilled
in the art in practicing the present invention. Even so, this detailed
description should not be construed to unduly limit the present
invention as modifications and variations in the the embodiments
discussed herein can be made by those of ordinary skill in the art
without departing from the spirit or scope of the present inventive
discovery.
All publications, patents, patent applications and other references
cited in this application are herein incorporated by reference in
their entirety as if each individual publication, patent, patent
application or other reference were specifically and individually
indicated to be incorporated by reference.
As used herein, the term "animal" includes, but is not
limited to, mammals, including human beings. It should be noted
that the complexes and methods disclosed herein are applicable in
both human and veterinary medicine. Thus, the present compounds
and methods can be applied to humans, domestic pets such as cats,
dogs, rodents, birds etc., farm animals such as cows, sheep, goats,
pigs, horses, etc., zoo animals, etc.
Amino acids are indicated herein using the single letter notation
conventional in the art. When used in amino acid sequences, the
letter "x" designates any amino acid. When used in an
amino acid sequence, a "/" between two adjacent letters
indicates that either of the amino acids listed can be used. When
used in nucleotide sequences, the letter "n" designates
A, T, C or G. Except as noted in Table 2, the use of upper or lowercase
letters to define the amino acids in a sequence is not meant to
convey a particular stereospecificity to the acids within the sequence.
Structure of Membrane-Permeant Peptide Covalent and Coordination
Complexes
The general structure of the present invention compounds comprises
a unique combination of peptide components to produce a new class
of imaging and therapeutic conjugates that will enable interrogation
of, and/or interaction with, the desired intracellular processes
within living cells in the whole organism. This novel class of agents
in its simplest form comprises three components: 1) a cell membrane-permeant
peptide sequence made up of D-amino acids, L-amino acids or a combination
of D- and L-amino acids; 2) a functional or non-functional linker
motif; and 3) a chelator moiety able to coordinate metals useful
in medical imaging and therapy (FIG. 1), or other cargo molecule
such as a diagnostic substance or pharmaceutically active agent.
The HIV-1 Tat basic peptide sequence is an example of the prototypic
cell membrane-permeant component. The linker region can comprise
amino acid residues, or substituted or unsubstituted hydrocarbon
chains useful for connecting the Tat peptide and the metal chelator,
for example, via peptide bonds. The linker region can be designed
to be non-functional or functional. "Non-functional" refers
to non-reactive hydrocarbon chains, simple amino acid sequences,
or other sequences that simply bind covalently to the Tat peptide
residues on one end and the cargo molecule on the other end. A "functional
linker" can comprise amino acid residues that confer biological
properties useful for imaging, diagnostics, therapy, etc. Such a
functionality could include peptide or protein binding motifs, protein
kinase consensus sequences, protein phosphatase consensus sequences,
or protease-reactive or protease-specific sequences. Protease sequences
are particularly useful as they will result in amplification of
an imaging, radiotherapeutic, diagnostic, or therapeutic effect
through enzymatic action on the conjugate complex, thereby increasing
the intracellular concentration of a cleaved and subsequently trapped
metal-chelate or other cargo molecule.
Cell Membrane-Permeant Peptides
The cell membrane-permeant basic peptide component of the complexes
of the present invention can comprise any amino acid sequence that
confers the desired intracellular translocation and targeting properties
to the covalent or coordination complexes. Preferably, these amino
acid sequences are characterized by their ability to confer transmembrane
translocation and internalization of a complex construct when administered
to the external surface of an intact cell, tissue or organ. The
complex would be localized within cytoplasmic and/or nuclear compartments
as demonstrated by a variety of detection methods such as, for example,
fluorescence microscopy, confocal microscopy, electron microscopy,
autoradiography, or immunohistochemistry.
Cell membrane-permeant peptide sequences useful in practicing the
present invention include, but are not limited to, RQARRNRRRRWRERQR-51
(HIV-1 Rev protein basic motif; SEQ ID NO: 1); MPKTRRRPRRSQRKRPPTP-119
(HTLV-1 Rex protein basic motif; SEQ ID NO: 2) (Kubota et al., Biochem.
Biophys. Res. Comm., 162:963-970, 1989); the third helix of the
homeodomain of Antennapedia (Derossi, et al., J. Biol. Chem. 271:18188-93,
1996) (43-RQILIWFQNRRMKWLL-58 (SEQ ID NO: 3)); a peptide derivable
from the heavy chain variable region of an anti-DNA monoclonal antibody
(Avrameas, et al., Proc. Natl. Acad. Sci. 95:5601-06, 1998) (VAYISRGGVSTYYSDTVKGRFTRQKYNKRA
(SEQ ID NO: 4)); and the Herpes simplex virus VP22 protein (Elliot
and O'Hare, Cell, 88:223-33, 1997) (1-MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRG
EVRFVQYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPA GSGGAGRTPTTAPRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPA
SRAPTVQLWQMSRPRTDEDLNELLGITHRVTVCEGKNLLQRANELVNPDVV QDVDAATATRGRSAASRPTERPRAPARSASRPRRPVE-246
(SEQ ID NO: 5)). In a preferred embodiment, the basic peptide is
derivable from the human immunodeficiency virus type 1 (HIV-1) Tat
protein (Fawell et al., Proc. Natl. Acad. Sci., 91:664-68, 1994).
In particular, the Tat peptide can comprise any sequential residues
of the Tat protein basic peptide motif 37-72 (Vives et al., J. Biol.
Chem., 272:16010-16017, 1997) (37-CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ-72
(SEQ ID NO: 6).
Preferred examples of conjugate sequences with favorable cell uptake
and U/W ratios include arginine-rich permeation peptide sequences
based on the Tat basic peptide, such as: acetyl-RKKRRNRRR-AHA-.epsilon.KGC-amide
(SEQ ID NO: 33); acetyl-RKKRROrnRRR-AHA-.epsilon.KGC-amide (SEQ
ID NO: 34); acetyl-RKKRRERRR-AHA-.epsilon.KGC-amide (SEQ ID NO:
35); and acetyl-RKKRRNorleuRRR-AHA-.epsilon.KGC-amide (SEQ ID NO:
36) where Orn is ornithine and Norleu is norleucine.
Other permeant peptides useful in the present invention include
poly-Arg, RRRRRRRRR (SEQ ID NO: 37); amphipathic polycationic peptide,
RAARRAARR (SEQ ID NO: 38); and the viral permeation peptide, PLSSIFSRIGDP
(SEQ ID NO: 39). As with all the inventive permeation peptide sequences,
such sequences may contain and shall be understood to encompass,
the variable N-terminus, -4 substitutions and other modifications
taught herein.
The minimum number of amino acid residues can be in the range of
from about three to about nine, preferably from about three to about
five, and most preferably about four, i.e., the minimal requirement
for one alpha helical turn. A preferred embodiment comprises Tat
protein residues 48-57 (GRKKRRQRRR) (SEQ ID NO: 7). Residue number
may be selected or modified to achieve a desired level of cellular
uptake as there is a correlation between decreased length of at
least some permeation peptides and decrease cellular uptake of the
conjugate. For example, to generate the sequences identified as
13a,14a,15,16,17 of Table 2, one additional amino acid was removed
from the N-terminus of the longest Tat basic domain sequence (RKKRRQRRR)
while all other aspects of the peptide remained the same. From this
data, a correlation between decreasing length and decreasing uptake
of Tat basic domain peptide was observed (FIG. 11). Similarly, there
was an overall decrease in net cell uptake of the L-poly-Arg peptide
as the length shortened from poly-Arg.sub.9 to poly-Arg.sub.7 and
of D-poly-Arg peptide as length shortened from poly-Arg.sub.8 to
poly-Arg.sub.6. However, for the series 18-21 (RAARRAARR), a putative
amphipathic sequence with .alpha.-helical properties, there was
relatively modest uptake and no change with decreasing length (FIG.
11).
In one preferred embodiment any of the aforementioned membrane
peptides may contain at least one D-amino acid. In another preferred
embodiment, a majority of the amino acid residues in any of the
aforementioned peptides can comprise D-amino acids. In yet another
preferred embodiment, any of the aforementioned peptides are comprised
entirely of D-amino acids in forward sequence or inverse sequence
(retro-inverse). In another preferred embodiment, all the amino
acids of the membrane permeant peptide are D-amino acids whereas
the remaining amino acids in the conjugate, including the chelation
moiety, may be either D or L enantiomers. This aspect of the invention
arises from the surprising discovery that altering the chirality
of the chelation moiety to all D-amino acids showed no significant
difference in uptake compared to the L-peptides.
As used herein, the term "amino acid" is applicable not
only to cell membrane-permeant peptides, but also to linker moieties,
coordination ligands, and other cargos, including pharmaceutical
agents, i.e., all the individual components of the present complexes.
The term "amino acid" is used in its broadest sense, and
includes naturally occurring amino acids as well as non-naturally
occurring amino acids, including amino acid analogs and derivatives.
The latter includes molecules containing an amino acid moiety. One
skilled in the art will recognize, in view of this broad definition,
that reference herein to an amino acid includes, for example, naturally
occurring proteogenic L-amino acids; D-amino acids; chemically modified
amino acids such as amino acid analogs and derivatives, including
.beta.-amino acids; naturally occurring non-proteogenic amino acids
such as norleucine, .beta.-alanine, ornithine, etc.; and chemically
synthesized compounds having properties known in the art to be characteristic
of amino acids. As used herein, the term "proteogenic"
indicates that the amino acid can be incorporated into a peptide,
polypeptide, or protein in a cell through a metabolic pathway.
The incorporation of non-natural amino acids, including synthetic
non-native amino acids, substituted amino acids, or one or more
D-amino acids into the peptides (or other components of the complexes)
of the present invention (subsequently referred to herein as "D-peptides")
is advantageous in a number of different ways. D-amino acid-containing
peptides exhibit increased stability in vitro or in vivo compared
to L-amino acid-containing counterparts. Thus, the construction
of peptides incorporating D-amino acids can be particularly useful
when greater intracellular stability is desired or required. More
specifically, D-peptides are resistant to endogenous peptidases
and proteases, thereby providing better oral transepithelial and
transdermal delivery of linked drugs and conjugates, improved bioavailability
of membrane-permeant complexes, and prolonged intravascular and
interstitial lifetimes when such properties are desirable. The use
of D-peptides can also enhance transdermal and oral transepithelial
delivery of linked drugs and other cargo molecules. As shown in
Example 14, the use of D-amino acids in the membrane permeant peptide
greatly increases the accumulation of linked drugs or other cargo
molecules into cells. Additionally, D-peptides cannot be processed
efficiently for major histocompatibility complex class II-restricted
presentation to T helper cells, and are therefore less likely to
induce humoral immune responses in the whole organism. Peptide conjugates
can therefore be constructed using, for example, D-peptide membrane
permeant sequences, L-peptide functional linker domains, and D-peptide
chelation sequences. In this embodiment, only the functional L-peptide
linker region would be able to interact with native enzymatic activities
such as proteases, kinases, and phosphatases, thereby providing
enhanced selectivity, prolonged biological half-life, and improved
signal-to-noise ratio for selected imaging applications. On the
other hand, when it is more desirable to allow the peptide to remain
active for only a short period of time, the use of L-amino acids
in the peptide can allow endogenous peptidases in a cell to digest
the peptide in vivo, thereby limiting the cell's exposure to the
membrane-permeant peptide covalent and coordination complexes comprising
the peptides disclosed herein. It will be apparent that it is possible
to construct complexes in which different portions contain either
D- or L-amino acids. For example and without limitation, it is possible
to construct a complex in which a cell permeant peptide and a metal
chelator comprised of D-amino acids are connected by a functional
linker comprised of L-amino acids. Other such combinations will
be readily apparent to those of ordinary skill in the art and are
within the scope of the present invention.
In addition to using D-amino acids, those of ordinary skill in
the art are aware that modifications in the amino acid sequence
of a peptide, polypeptide, or protein can result in equivalent,
or possibly improved, second generation peptides, etc., that display
equivalent or superior functional characteristics when compared
to the original amino acid sequence. The present invention accordingly
encompasses such modified amino acid sequences. Alterations can
include amino acid insertions, deletions, substitutions, truncations,
fusions, inversions, shuffling of subunit sequences, and the like,
provided that the peptide sequences produced by such modifications
have substantially the same functional properties as the naturally
occurring counterpart sequences disclosed herein. Thus, for example,
modified cell membrane-permeant peptides should possess substantially
the same transmembrane translocation and internalization properties
as the naturally occurring counterpart sequence.
One factor that can be considered in making such changes is the
hydropathic index of amino acids. The importance of the hydropathic
amino acid index in conferring interactive biological function on
a protein has been discussed by Kyte and Doolittle (J. Mol. Biol.,
157: 105-132, 1982). It is accepted that the relative hydropathic
character of amino acids contributes to the secondary structure
of the resultant protein. This, in turn, affects the interaction
of the protein with molecules such as enzymes, substrates, receptors,
DNA, antibodies, antigens, etc.
Based on its hydrophobicity and charge characteristics, each amino
acid has been assigned a hydropathic index as follows: isoleucine
(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate/glutamine/aspartate/asparagine
(-3.5); lysine (-3.9); and arginine (-4.5).
As is known in the art, certain amino acids in a peptide or protein
can be substituted for other amino acids having a similar hydropathic
index or score and produce a resultant peptide or protein having
similar biological activity, i.e., which still retains biological
functionality. In making such changes, it is preferable that amino
acids having hydropathic indices within .+-.2 are substituted for
one another. More preferred substitutions are those wherein the
amino acids have hydropathic indices within .+-.1. Most preferred
substitutions are those wherein the amino acids have hydropathic
indices within .+-.0.5.
Like amino acids can also be substituted on the basis of hydrophilicity.
U.S. Pat. No. 4,554,101 discloses that the greatest local average
hydrophilicity of a protein, as governed by the hydrophilicity of
its adjacent amino acids, correlates with a biological property
of the protein. The following hydrophilicity values have been assigned
to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0.+-.1);
serine (+0.3); asparagine/glutamine (+0.2); glycine (0); threonine
(-0.4); proline (-0.5.+-.1); alanine/histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8);
tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). Thus,
one amino acid in a peptide, polypeptide, or protein can be substituted
by another amino acid having a similar hydrophilicity score and
still produce a resultant protein having similar biological activity,
i.e., still retaining correct biological function. In making such
changes, amino acids having hydropathic indices within .+-.2 are
preferably substituted for one another, those within .+-.1 are more
preferred, and those within .+-.0.5 are most preferred.
As outlined above, amino acid substitutions in the peptides of
the present invention can be based on the relative similarity of
the amino acid side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, etc. Exemplary substitutions that
take various of the foregoing characteristics into consideration
in order to produce conservative amino acid changes resulting in
silent changes within the present peptides, etc., can be selected
from other members of the class to which the naturally occurring
amino acid belongs. Amino acids can be divided into the following
four groups: (1) acidic amino acids; (2) basic amino acids; (3)
neutral polar amino acids; and (4) neutral non-polar amino acids.
Representative amino acids within these various groups include,
but are not limited to: (1) acidic (negatively charged) amino acids
such as aspartic acid and glutamic acid; (2) basic (positively charged)
amino acids such as arginine, histidine, and lysine; (3) neutral
polar amino acids such as glycine, serine, threonine, cysteine,
cystine, tyrosine, asparagine, and glutamine; and (4) neutral non-polar
amino acids such as alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine. It should be noted that
changes which are not expected to be advantageous can also be useful
if these result in the production of functional sequences.
Additionally, substitutions may be made based on sequence specific
effects and the charge of particular amino acids. For example, it
is of particular usefulness in the present invention to increase
the cationic charge of the permeation peptide used in the conjugate
to enhance cellular uptake. One method of accomplishing this is
the substitution of one or more positively charged amino acids for
one or more negatively charged acids in the permeant peptide. For
example, substitution of the positively charged amino acid Orn for
the naturally occurring negatively charged amino acid at C-4 in
the Tat basic peptide sequence increases the cellular uptake of
a conjugate comprising such peptide (FIG. 12). On the other hand,
substituting at the same position with the negatively charged Glu,
decreased cellular uptake.
The permeation peptide sequences of the present invention are effective
regardless of N-terminus biotinylation or acetylation. Specifically,
the presence of biotin or acetyl groups on the N-terminus of the
various permeation peptides did not significantly change their cell
uptake as shown in Table 2. Thus, sequence identifications herein
which include specific N-terminus moieties should not be interpreted
as requiring any N-terminus or as limiting such sequences to such
moieties.
Since small peptides can be easily produced by conventional solid
phase synthetic techniques, the present invention includes peptides,
linker regions, and cargo molecules such as those discussed herein,
containing the amino acid modifications discussed above, alone or
in various combinations. To the extent that such modifications can
be made while substantially retaining the cell membrane permeant
and targeting properties of the peptide, and the biological function
and specificity of the linker region and cargo moieties, they are
included within the scope of the present invention. The utility
of such modified peptides, linkers, and cargos can be determined
without undue experimentation by, for example, the methods described
in the examples below.
Linker Regions
Linker regions useful in linking the Tat or other cell membrane-permeant
peptides described herein and cargos such as drugs or diagnostic
substances such as metal chelator moieties can comprise amino acid
residues or substituted or unsubstituted hydrocarbon chains. Useful
linker regions include natural and unnatural biopolymers. Examples
of natural linkers include oligonucleotides and L-oligopeptides,
while examples of unnatural linkers are D-oligopeptides, lipid oligomers,
liposaccharide oligomers, peptide nucleic acid oligomers, polylactate,
polyethylene glycol, cyclodextrin, polymethacrylate, gelatin, and
oligourea (Schilsky, et al., Eds., Principles of Antineoplastic
Drug Development and Pharmacology, Marcel Dekker, Inc., New York,
1996, pp. 741). The linker region can be designed to be functional
or non-functional.
"Non-functional" as applied to linker regions means any
non-reactive amino acid sequence, hydrocarbon chain, etc., that
can bond covalently to Tat or other cell membrane-permeant peptide
residues on one end and a drug or chelating ligand, for example,
on the other end. As used herein, the term "non-reactive"
refers to a linker that is biologically inert and biologically stable
when a complex containing the linker is contacted by cells or tissues.
Upon characterization, the linker and conjugate can be shown to
remain intact as the parent compound when analyzed by reverse phase
HPLC or TLC. Non-functional linkers are desirable in the design
and synthesis of complexes useful, for example, in non-specific
labeling of white blood cells for imaging infections, in non-specific
labeling of tissues for perfusion imaging, and in interaction with
any intracellular receptor or other activity or site. Examples of
non-functional linkers include, but are not limited to, amino hexanoic
acid, glycine, alanine, or short peptide chains of nonpolar amino
acids such as di- or tri-glycine or tri-alanine. Hydrocarbon chain
linkers can include both unsubstituted and substituted alkyl, aryl,
or macrocyclic R groups, as disclosed in U.S. Pat. No. 5,403,574.
R groups are found in the general formula --CR.sub.3 where R can
be identical or different and includes the elements H, C, N, O,
S, F, Cl, Br, and I. Representative examples include, but are not
limited to, --CH.sub.3, --CH.sub.2CH.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(CH.sub.3).sub.2OCOCH.sub.3, CONH.sub.2, --C.sub.6H.sub.5,
--CH.sub.2(C.sub.6H.sub.4)OH, or any of their isomeric forms. "Alkyl"
is intended to mean any straight, branched, saturated, unsaturated
or cyclic C.sub.1-20 alkyl group. Typical C.sub.1-C.sub.20 alkyl
groups include, but are not limited to, methyl, ethyl, n-propyl,
i-propyl, n-butyl, t-butyl, i-butyl, pentyl and hexyl groups. "Aryl"
is intended to mean any aromatic cyclic hydrocarbon based on a six-membered
ring. Typical aryl groups include, but are not limited to, phenyl,
naphthyl, benzyl, phenethyl, phenanthryl, and anthracyl groups.
The term "macrocycle" refers to R groups containing at
least one ring containing more than seven carbon atoms. "Substituted"
is intended to mean any alkyl, aryl or macrocyclic groups in which
at least one carbon atom is covalently bonded to any functional
groups comprising the atoms H, C, N, O, S, F, Cl, Br or I.
"Functional" as applied to linker regions means, for
example, amino acid residues, oligonucleotides, oligosaccharides,
peptide nucleic acids, or substituted or unsubstituted hydrocarbon
chains as discussed above that confer biological or physicochemical
properties useful for the practice of this invention when incorporated
into the linker component. Such properties include, for example,
utility in medical imaging, radiotherapy, diagnosis, and pharmacological
treatment of disease states by virtue of interaction of the functional
linker region with intracellular components, which can be unique
to, or highly characteristic of, cells in particular physiological
or disease states. Such interaction can include, for example, binding
or other reaction, for example cleavage, of the functional linker
region due to interaction with intracellular components. However
this interaction occurs, such interaction results in selective retention
of the cargo molecule within particular cells due to the presence
of a particular intracellular component(s) within such cells. The
interaction of the functional linker with the intracellular component
thereby confers target cell specificity to a peptide complex containing
a particular functional linker moiety. Examples of functional linkers
are peptide or protein binding motifs, protein kinase consensus
sequences, protein phosphatase consensus sequences, or protease-reactive
or protease-specific sequences. Additional examples include recognition
motifs of exo- and endo-peptidases, extracellular metalloproteases,
lysosomal proteases such as the cathepsins (cathepsin B), HIV proteases,
as well as secretases, transferases, hydrolases, isomerases, ligases,
oxidoreductases, esterases, glycosidases, phospholipases, endonucleases,
ribonucleases and .beta.-lactamases.
Specific examples of useful consensus sequences and recognition
motifs are: 14-3-3 protein binding motifs such as RSXSphosphoSXP
(SEQ ID NO: 8) or RXY/FXphosphoSXP (SEQ ID NO: 9) (Yaffe et al.,
Cell, 91:961-971, 1997). Preferred embodiments include the 14-3-3
protein binding motifs RLSHphosphoSLP (SEQ ID NO: 10), RLYHphosphoSLP
(SEQ ID NO: 11) (Peng, et al., Science 277:1501-1505, 1997); and
RLSHphosphoSLG (SEQ ID NO: 12). Protease-reactive or specific consensus
sequences include, for example, those peptide sequences recognized
by interleukin-1.beta. converting enzyme (ICE) homologues, such
as caspase-1, CPP32/Yama/apopain/caspase-3, NEDD2/Ich-1/caspase-2,
TX/Ich-2/caspase-4, ICE-LAP3/MCH-3/CMH-1/caspase-7, ICE-LAP6/caspase-9,
and FLICE/MACH/caspase-8 ((Nakagawara et al. Cancer Res., 57:4578-4584,
1997) and references therein), including YEVDx (SEQ ID NO: 13) for
Caspase-1, YDVADx (SEQ ID NO: 14) for Caspase-2, DEVDx (SEQ ID NO:
15) and DMQDx (SEQ ID NO: 16) for Caspase-3, LEVDx ((SEQ ID NO:
17) for Caspase-4, VEIDx (SEQ ID NO: 18) for Caspase-6, DEVDx (SEQ
ID NO: 19) for Caspase-7, IETDx (SEQ ID NO: 20) for Caspase-8, and
IEADx (SEQ ID NO: 21) for Caspase-10 (Villa, et al., Trends Biochem
Sci 22:388-393, 1997); SQVSQNY-PIVQNLQ (SEQ ID NO: 22) for the HIV
p17-p24 A cleavage site, and CTERQAN-FLGKIWP (SEQ ID NO: 23) for
the HIV p7-p1 D cleavage site (Ratner, et al., Nature 313:277-284,
1985; Welch, et al., Proc Natl Acad Sci USA 88:10792-10796, 1991);
xR(R/K)x(S/T)x for Protein Kinase A, x(R/K).sub.2-3x(S/T)x for Protein
Kinase G, X(R/K.sub.1-3,x.sub.0-2)(S/T)(X.sub.0-2,R/K.sub.1-3)x
for Protein Kinase C, xRxx(S/T)x for Calmodulin Kinase II, KRKQI(S/T)VR
(SEQ ID NO: 24) for Phosphorylase b Kinase, TRDIYETDYYRK (SEQ ID
NO: 25) for Insulin Receptor Kinase, and TAENAEYLRVAP (SEQ ID NO:
26) for EGF Receptor Kinase (Kemp and Pearson, Trends Biochem Sci
15:342-346, 1990; Kennelly and Krebs, J Biol Chem 266:15555-15558,
1991). Examples of other useful non-peptide motifs include, for
example, DNA recognition sequences such as 3'-TCTTGTnnnACAAGA-5'
(SEQ ID NO: 27) for the glucocorticoid hormone response element,
3'-TCCAGTnnnACTGGA-5' (SEQ ID NO: 28) for the estrogen receptor
response element, and 3'-TCCAGTACTGGA-5' (SEQ ID NO: 29) for the
thyroid hormone response element (Fuller, FASEB J 5:3092-3099, 1991).
Additional sequences known to those skilled in the art and available
by reference to public databases can be incorporated into the linker
moieties of the present complexes. Well known protein, DNA, and
RNA databases available to investigators working in the art of biomedical
and pharmaceutical sciences include those linked to the U.S. National
Institutes of Health Web Site, such as: http://molbio.info.nih.gov/molbio/,
all herein incorporated by reference. A biomolecule or fragment
thereof containing a putative recognition motif can be identified
by sequence comparison of the primary structure with a primary consensus
sequence or individual sequence of a protein or biomolecule in the
databases using routine computerized sequence scanning methods such
as, for example, BLAST.
When incorporated into the intact Tat or other peptide complexes
of the present invention, such sequence motifs will be acted on
solely or selectively in those cells containing the appropriate
intracellular sequence-specific or sequence-reactive protein, which
will alter the intracellular/subcellular distribution and retention
of the cargo molecule, e.g., a drug or metal chelate. For example,
protease sequences are particularly useful as they result in enzymatic
amplification of an imaging or radiotherapeutic effect through enzymatic
action on the conjugate complex, thereby cleaving and subsequently
trapping metal-chelates within intracellular compartments, leading
to an increase in the concentration of the metal-complex fragment.
To further illustrate this principle, if the intracellular target
to be detected is a specific protease activity of the caspase family,
then when a coordination complex of the present invention comprising
the components (Tat peptide)-(caspase-3 motif linker)-(chelate{metal})
translocates into a cell containing caspase-3, the enzyme will cleave
the complex in the linker region, thereby releasing the metal-chelate
within the cell interior, which can then be monitored by conventional
techniques. Of course, such target specificity could also be accomplished
by the use of a caspase reactive diagnostic substance as well.
Cells or tissues having other biological, biochemical, or physiological
activities can also be detected when the appropriate functional
linker is incorporated into the covalent or coordination complex.
For example, a hexose sequence recognized by .beta.-galactosidase
can be synthesized into the linker region of the invention compounds,
e.g., as (Tat peptide)-(D-galactose-D-glucose)-(chelate{metal}).
Then, upon administration to cells transduced with a marker gene
that encodes .beta.-galactosidase, for example in gene therapy,
only those cells which express .beta.-galactosidase will cleave
and retain the chelate-metal complex for subsequent detection by
external imaging devices.
Metal-chelate moieties can be synthesized to possess net charge,
for example, by substitution of K for G on the .epsilon.KGC chelation
peptide as illustrated in Example 1. This is useful for in vivo
applications in a whole animal. Because non-targeted or unreacted
Tat peptide conjugates are capable of bidirectionally translocating
across membranes, as the extracellular concentration of a Tat peptide
conjugate declines, the intracellular intact Tat peptide conjugate
will translocate outwardly and be cleared from the animal via the
bloodstream. However, where protease cleavage acts on the peptide,
the Tat fragment is separated from the chelate fragment, which further
generates a positive charge at the amino-terminus of the cleaved
chelate fragment. Thus, the overall charge of the released peptide
chelate complex will be polycationic. This cluster of charge combined
with the lack of an attached Tat permeation sequence will render
the cleaved chelate fragment impermeant to the cell membrane, in
effect trapping the chelate fragment within the cell both in vivo
and in vitro. In cells lacking the targeted protease activity, the
intact Tat peptide-chelate complex translocates outwardly into the
extracellular spaces as the extracellular concentration of the Tat
peptide decreases. This clearance has been found to occur surprisingly
rapidly in vivo. The present invention exploits this high clearance
rate to provide high target-to-background ratios for imaging, diagnostics,
and therapeutic delivery of metal chelates and drug conjugates to
specific cells, tissues and organs.
In cases where the metal-chelate comprises a radioactive metal,
then external imaging devices such as scintigraphic gamma cameras
or SPECT will only detect high radioactivity within cells, tissues
or organs containing the desired biological activity. In contrast,
if the metal-chelate comprises a ligand complexed with a relaxivity
metal, such as Gd-DTPA, then the resulting enhanced T1 relaxivity
would be detectable within cells and tissues of living patients
using appropriate T1-weighted pulse sequences generated by clinical
magnetic resonance imaging (MRI) devices. Those skilled in the art
can readily operate the appropriate MRI device to detect proton
relaxivity changes in bodily water induced by relaxivity complexes
known as MR contrast agents (Stark and Bradley, Magnetic Resonance
Imaging, C. V. Mosby Co., St. Louis, 1988, pp. 1516). Thus, the
present invention overcomes a limitation present in existing methods,
which do not provide for the intracellular deposition of peptide
chelate-metal complexes for targeted medical imaging with SPECT/PET
and radiotherapeutic applications, nor allow the interrogation of
changes in intracellular proton relaxivity with MRI devices. In
contrast, the present invention provides for the intracellular delivery
and targeted retention of desired metal complexes.
Various chelation peptides may be used in the present invention
to ensure effective chelation, to enhance cell uptake of the conjugate
and to meet other structural or functional goals of a particular
conjugation strategy. For example, the Lys-Gly-Cys utilized in most
of the exemplar conjugates was selected in light of its ability
to efficiently chelate .sup.99mTc. Using a His-Gly chelation peptide
to chelate .sup.99mTc(CO).sub.3 showed a significant increase in
uptake of the conjugate. The His-Gly peptide would also allow for
radiolabelling of the N-terminus and further conjugation at the
C-terminus via an additional Cys amino acid. Using a Gly-Lys chelation
peptide along with orthogonal conjugation of the chelation cargo
to the e-amine of the Lys results in significant reduction in conjugate
uptake but allowed double or triple labeling of peptides.
Other variations are possible wherein the Tat or other peptide-linker-metal
complexes contain a functional linker and are sufficiently stable
to be delivered to the desired cells and translocated into the cell
interior, where they will be acted upon by the targeted intracellular
biochemical activity and the retained metal-chelates detected with
imaging devices as above.
In addition to radioactive and non-radioactive metals, pharmacologically
active substances, prodrugs, cytotoxic substances, and diagnostic
substances such as fluorochromes, dyes, enzyme substrates, etc.,
can be coupled to the linkers of the present membrane-permeant peptide
complexes. A wide variety of drugs are suitable for use with the
present invention, and include, for example, conventional chemotherapeutics,
such as vinblastine, doxorubicin, bleomycin, methotrexate, 5-fluorouricil,
6-thioguanine, cytarabine, cyclophosphamide, taxol, taxotere, cis-platin,
adriamycin, mitomycin, and vincristine as well as other conventional
chemotherapeutics as described in Cancer: Principles and Practice
of Oncology, 5th Ed., V. T. Devita, S. Hellman, S. A. Rosenberg,
J. B. Lippincott, Co., Phila, 1997, pp. 3125. Also suitable for
use in the present invention are experimental drugs, such as UCN-01,
acivicin, 9-aminocamptothecin, azacitidine, bromodeoxyuridine, bryostatin,
carboplatin, dideoxyinosine, echinomycin, fazarabine, hepsulfam,
homoharringtonine, iododeoxyuridine, leucovorin, merbarone, misonidazole,
pentostatin, semustine, suramine, mephthalamidine, teroxirone, triciribine
phosphate and trimetrexate as well as others as listed in NCI Investigational
Drugs, Pharmaceutical Data 1994, NIH Publications No. 94-2141, revised
January 1994.
In addition, the radioactive and non-radioactive metals, pharmacologically
active substances, prodrugs, cytotoxic substances, and diagnostic
substances used herein may themselves provide target cell specificity.
Such specificity may be particularly effective where such substances
are used in a conjugate with a non-functional linker of the present
invention.
Other useful drugs include anti-inflammatories such as Celebrex,
indomethacin, flurbiprofen, ketoprofen, ibuprofen and phenylbutazone;
antibiotics such as beta-lactams, aminoglycosides, macrolides, tetracyclines,
pryridonecarboxylic acids and phosphomycin; amino acids such as
ascorbic acid and N-acetyltryptophan; antifungal agents; prostaglandins;
vitamins; steroids; and antiviral agents such as AZT, DDI, acyclovir,
gancyclovir, idoxuridine, amantadine and vidarabine.
Pharmacologically active substances that can be conjugated to the
complexes of the present invention include, but are not limited
to, enzymes such as transferases, hydrolyses, isomerases, proteases,
ligases, kinases, and oxidoreductases such as esterases, phosphatases,
glycosidases, and peptidases; enzyme inhibitors such as leupeptin,
chymostatin and pepstatin; growth factors; and transcription factors
or domains derived from each.
In addition, the present invention can be used to deliver fluorochromes
and vital dyes into cells. Examples of such fluorochromes and vital
dyes are well known to those skilled in the art and include, for
example, fluorescein, rhodamine, coumarin, indocyanine Cy 5.5, NN382,
Texas red, DAPI, EDANS, DABCYL and ethidium bromide.
The delivery of drug and pharmacologically active compounds into
the cell interior can be enhanced by direct conjugation to the Tat
or other membrane-permeant peptides of the present invention. The
coupling of such compounds to a functional linker placed between
a D-amino acid containing cell membrane-permeant peptide and the
active agent, thereby enabling enhanced, functionally selective,
intracellular trapping of the drug or drug conjugate, is new. A
drug or prodrug conjugate designed as described herein would enable
selective delivery (and retention) of bioactive agents and therapeutic
or biologic enhancers useful in therapy including, but not limited
to, granulocyte-stimulating factors, platelet-stimulating factors,
erythrocyte-stimulating factors, macrophage-colony stimulating factors,
interleukins, tumor necrosis factors, interferons, other cytokines,
monoclonal antibodies, immune adjuvants and gene therapy vectors
(Devita, et al., Biologic Therapy of Cancer, 2nd Ed., J. B. Lippincott,
Co., Phila, 1995, pp. 919), and drugs into the cell interior in
a manner analogous to the selective trapping of metal chelates as
described above. Linker functionality can include any motif that
can be acted on by a specific intracellular agent, such as the enzymes
discussed above, or ribozymes, for example. Examples of such linker
functionalities include low molecular weight peptide or protein
binding motifs, protein kinase consensus sequences, protein phosphatase
consensus sequences, or protease-specific sequences. As explained
previously, protease-reactive or protease-specific sequences are
particularly useful in that amplification of the therapeutic effect
would occur through enzymatic action on the linker region of the
drug or prodrug conjugate, thereby releasing the pharmacological
agent in the cell cytosol, and increasing the intracellular retention
and concentration of the agent.
Pharmacologically active substances, cytotoxic substances, diagnostic
substances, etc., can be coupled to the appropriate cell membrane-permeant
peptide-linker conjugate through either the amino- or carboxy-terminus
of the linker region in a manner analogous to that described in
Example 1. For example, drug conjugates wherein the carboxy-terminus
of the peptide linker is coupled to a bioactive substance can be
prepared by the use of an active ester of the desired bioactive
substance in the presence of a dehydrating agent. Examples of active
esters that can be used in the practice of the present invention
include the hemi-succinate esters of N-hydroxysuccinimide, sulfo-N-hydroxy-succinimide,
hydroxybenzotriazole, and p-nitrophenol. Dehydration agents include
dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(ECD), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide
(EDCI). The use of ECD to form conjugates is disclosed in U.S. Pat.
No. 4,526,714, the disclosure of which is fully incorporated by
reference herein. Other examples of coupling reagents include glutathione,
3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT),
onium salt-based coupling reagents, polyoxyethylene-based heterobifunctional
cross-linking reagents, and other reagents that facilitate the coupling
of organic drugs and peptides to various ligands (Haitao, et al.,
Organ Lett 1:91-94, 1999; Albericio, et al., J Organic Chemistry
63:9678-9683, 1998; Arpicco, et al., Bioconjugate Chem 8:327-337,
1997; Frisch, et al., Bioconjugate Chem 7:180-186, 1996; Deguchi,
et al., Bioconjugate Chem 10:32-37, 1998; Beyer, et al., J Med Chem
41: 2701-2708, 1998; Dirven, et al., Chem Res Toxicol 9:351-360,
1996; Drouillat, et al., J Pharm Sci 87:25-30, 1998; Trimble, et
al., Bioconjugate Chem 8:416-423, 1997). Chemicals, reagents and
techniques useful in drug cross-linking and peptide conjugation
are disclosed in general texts well known to those skilled in the
art (Dawson, et al., (Eds.), Data for Biochemical Research, 3rd
Ed., Oxford University Press, Oxford, UK, 1986, pp. 580; King, (Ed.),
Medicinal Chemistry: Principles and Practice, Royal Society of Chemistry,
Cambridge, UK, 1994, pp. 313; Shan and Wong, (Eds.), Chemistry of
Protein Conjugation and Cross-Linking, CRC Press, Boca Raton, 1991,
pp. 328). Additional chemical coupling agents are described in U.S.
Pat. No. 5,747,641, hereby incorporated by reference in its entirety.
Conjugated Chelate Ligands and Drugs
The present invention also encompasses the use of chelation ligands
to form coordinate bonds with desired metals. The desired chelation
ligands are attached to the peptide conjugate where they bind radionuclides
and desired non-radioactive metals in a highly efficient and stable
manner. When the metal is a radionuclide, this allows the reporting
of the spatial location of the conjugate with external imaging devices
such as SPECT and PET detectors following administration of the
conjugate to an animal. As disclosed above, preferred embodiments
of the present invention permit the chelation moiety to be concentrated
within cellular and tissue compartments in proportion to specific
enzymatic or protein activities present in the cells therein. In
other preferred embodiments, where the metal is a selected therapeutic
radionuclide, the present invention allows the chelation moiety
to be concentrated within target cellular and tissue compartments
in proportion to a specific enzymatic or protein activity to deposit
radiation selectively within the target cell or tissue. In another
preferred embodiment, when the metal is a relaxivity metal, the
chelation moiety permits magnetic resonance imaging of the cell
or tissue. Alternatively, when the functional linker region of the
permeant peptide construct is conjugated to a drug, the drug will
be selectively deposited within the target cell or tissue by methods
of this invention.
Suitable chelation ligands are well known to those skilled in the
art and include, but are not limited to, diethylenetriaminepentaacetic
acid (DTPA), ethylenediaminetetraacetic acid (EDTA), tetraazacyclododecanetetraacetic
acid (DOTA), and other chelators that incorporate electron donating
atoms such as O, S, P or N as Lewis bases to bind the metal (Engelstad
and Wolf, "Contrast Agents", in Magnetic Resonance Imaging,
Stark and Bradley, Mosby, St. Louis, 1988, pp. 161-181). The present
complexes can also employ chelating ligands such as, but not restricted
to, those containing N.sub.2S.sub.2, N.sub.3S, N.sub.2SO and NS.sub.3)
moieties (Meegalla et al., J. Med. Chem., 40:9-17, 1997). Specific
examples (as shown below) wherein these chelation moieties are incorporated
into specific sequences of peptide residues, such as .epsilon.-amine
modified Lys-Gly-Cys tags, are especially convenient for synthesizing
the desired chelation groups directly into peptide-based sequences.
Preferred chelation ligands are peptides or modified peptides which
enable the chelation moiety to be incorporated into the peptide
construct directly by solid phase synthesis by use of appropriately
blocked peptide precursors compatible with commercial peptide synthesizers.
Examples of this preferred embodiment are illustrated below in more
detail. Alternatively, other preferred chelation ligands can be
chemically coupled to the peptide conjugate by use of one or more
of the linker reagents described above. Other preferred embodiments
of the invention encompass the conjugation of drugs or therapeutics,
including therapeutic peptides, to the functionalized linker region
attached to the permeant peptide. In one embodiment, the chelation
complexes of the present invention comprise a peptide-based chelator
wherein the coordination sites of the chelator are filled with a
metal useful in imaging or radiotherapy.
Radioactive and Non-Radioactive Metals
Useful metals for chelation into the complexes of the present invention
include radionuclides having decay properties that are amenable
for use as a diagnostic tracer or for deposition of medically useful
radiation within cells or tissues. The present invention consequently
encompasses the use of conjugated coordination complexes of a ligand
with a radioactive metal (radionuclide). The radioactive nuclide
can, for example, be selected from the group consisting of radioactive
isotopes of Tc, Ru, In, Ga, Co, Pt, Fe, Os, Ir, W, Re, Cr, Mo, Mn,
Ni, Rh, Pd, Nb, Cu and Ta, for example, Tc-99m, Tc-99, In-111, Ga-67,
Ga-68, Cu-64, Ru-97, Cr-51, Co-57, Re-188, and Re-186. Such complexes
can be used for medical imaging and specifically for SPECT or PET
imaging, as provided herein. Technetium-99m (Tc-99m; t1/2=6 hours;
140 keV emission photon) is the most commonly used radionuclide
in diagnostic nuclear medicine (Jurisson et al., Chem. Rev., 93:1137-156,
1993). It can be readily produced by molybdenum-99/technetium-99m
generators available in clinical nuclear medicine radiopharmacy
laboratories, and has favorable emission characteristics that enable
ready detection with clinical gamma cameras. While the complexes
of the present invention preferably contain Tc-99m and the closely
related rhenium isotopes (Re-186 and Re-188), other radionuclides
and metals, in addition to those already listed, useful for imaging
and radiotherapy such as I-123, I-125, I-130, I-131, I-133, Sc-47,
As-72, Se-72, Y-90, Y-88, Pd-100, Rh-100m, Sb-119, Ba-128, Hg-197,
At-211, Bi-212, Pd-212, Pd-109, Cu-67, Br-75, Br-76, Br-77, C-11,
N-13, O-15, F-18, Pb-203, Pb-212, Bi-212, Cu-64, Ru-97, Rh-105,
Au-198, and Ag-199 are also encompassed within the scope of this
invention. Moreover, the general availability of supplies of pertechnetate
from a variety of vendors makes it convenient to use kits for preparation
of various peptide complexes of Tc-99m. Labeling of the peptide
conjugates of the present invention with radioactive metals can
be readily performed. In preferred embodiments of this invention,
the peptide conjugate is radiolabeled with .sup.99mTc using standard
reducing agents with or without transmetallation reactions (Grummon,
et al., Inorg Chem 34:1764-1772, 1995; Lister-James, et al., J Nucl
Med 37:775-781, 1997; Meegalla, et al., J Med Chem 40:9-17, 1997).
Useful metals also include isotopes of those metals possessing
paramagnetism which produce water relaxation properties useful for
generating images with magnetic resonance imaging (MRI) devices.
Suitable relaxivity metals include, but are not limited to, Mn,
Cr, Fe, Gd, Eu, Dy, Ho, Cu, Co, Ni, Sm, Tb, Er, Tm, and Yb. Appropriate
chelation ligands to coordinate MR relaxivity metals can be readily
incorporated into the peptide complexes of this invention by the
methods previously described for radionuclides. Such chelation ligands
can include, but are not limited to, DTPA, EDTA, DOTA, TETA, EHPG,
HBED, ENBPI, ENBPA, and other macrocycles known to those skilled
in the art (Stark and Bradley, Magnetic Resonance Imaging, C. V.
Mosby Co., St Louis, 1988, pp 1516).
The peptide metal coordination complexes of the present invention
can be readily prepared by methods known in the art. For example,
a Tat or other cell membrane-permeant peptide conjugated to a linker
and a metal chelating moiety can be admixed with a salt of the radioactive
metal in the presence of a suitable reducing agent, if required,
in aqueous media at temperatures from room temperature to reflux
temperature, and the end-product coordination complex can be obtained
and isolated in high yield at both macro (carrier added, e.g., Tc-99)
concentrations and at tracer (no carrier added, e.g., Tc-99m) concentrations
(typically less than 10.sup.-6 molar). It is well established that
when (Tc-99m) pertechnetate (TcO.sub.4.sup.-) is reduced by a reducing
agent, such as stannous chloride, in the presence of chelating ligands
such as, but not restricted to, those containing N.sub.2S.sub.2,
N.sub.2SO, N.sub.3S and NS.sub.3 moieties, complexes of (TcO)N.sub.2S.sub.2,
(TcO)N.sub.2SO, (TcO)N.sub.3S and (TcO)NS.sub.3 are formed (Meegalla
et al. J. Med. Chem., 40:9-17, 1997). Another preferred method for
radio labeling the peptide involves the use of glucoheptonate together
with a reducing agent such as stannous chloride to label the chelation
moiety on the peptide (Lister-James, et al., J Nucl Med 37:775-781,
1997; Meegalla, et al., J Med Chem 40:9-17, 1997). Another preferred
labeling method involves one-step labeling of His-tagged peptides
with Tc(I)-carbonyl complexes (Waibel, et al., Nature Biotechnology,
17:897-901, 1999). Such Tc-99m labeling and chelating moieties can
be incorporated into potential receptor-selective imaging agents
(Horn and Katzenellenbogen, Nucl. Med. Biol., 24:485-498, 1997).
The incorporation of such moieties, specifically those that chelate
radioactive metals or other metals of interest for imaging (e.g.,
magnetic resonance relaxivity metals) or radiotherapy, into the
Tat or other peptide motif via the use of a functional linker, thereby
enabling selective intracellular delivery and retention of the metal
coordination complex, is new. Non-radioactive metals useful for
MR imaging can be incorporated into an appropriate chelator useful
for binding relaxivity metals which in turn has been conjugated
onto the peptide linker construct as described above. A preferred
embodiment of this invention is the coupling of DOTA to the peptide
conjugate using methods referenced above and using Gd as the MR
relaxivity metal. Gd can be chelated into the DOTA moiety by reaction
of chloride salts of Gd, such as GdCl.sub.3, with the peptide chelate
conjugate under mildly acidic conditions (pH 5-6) using standard
techniques (Stark and Bradley, Magnetic Resonance Imaging, C. V.
Mosby Co., St. Louis, 1988, pp. 1516; Wen-hong, et al., J Am Chem
Soc 121:1413-1414, 1999).
Other Applications
The present complexes can also be used in fluorescence resonance
energy transfer (FRET) to study intracellular processes. When used
with the FRET methodology, the functional linker is placed between
the fluorescent energy donor and acceptor. Examples of suitable
pairs of fluorescent energy donor and acceptors, as well as methods
for using FRET, are well known in the art and are described, for
example, in Ubarretxena-Belandia et al., Biochemistry, 38:7398-7405,
1999; Blomberg et al., Clin. Chem., 45:855-861, 1999; and Jamieson
et al., J Biol. Chem. 274:12346-12354, 1999. Near infrared fluorescent
(NIRF) probes may also be appended on each side of the linker such
that when the linker is intact, the probes are autoquenched and,
when the linker is specifically cleaved, the NIRF probes fluoresce
(Tyagi et al., Nature Biotech., 14:303-308, 1996).
In addition to providing compositions and methods for medical imaging,
other diagnostic methods, and drug delivery, the present invention
also provides methods for evaluating intracellular processes in
living cells in vivo and in tissues in vitro. Examples of such processes
include protein-protein binding, protein kinase activities, protein
phosphatase activities, or protease activities. Additional examples
include the activities of exo- and endo-peptidases, extracellular
metalloproteases, lysosomal proteases such as the cathepsins (cathepsin
B), as well as .alpha.-, .beta.-, and .gamma.-secretases, transferases,
hydrolases, isomerases, ligases, oxidoreductases, esterases, glycosidases,
phospholipases, endonucleases, ribonucleases and .beta.-lactamases
as they relate to the various disease states associated with loss
of function or gain of function for each. These methods are performed
by administering agents that are translocated across the plasma
membrane into cells and which are detectable in living cells despite
the presence of biological tissue intervening between the detection
device and the cells in their in situ location. Thus, cells in the
living body or in a tissue mass are detectable in situ.
In accordance with the present invention, living cells can be imaged.
Complexes of this invention useful in generating images are administered
to a patient, or to cells or a tissue specimen. Imaging procedures
include, but are not limited to, magnetic resonance imaging (MRI),
superconducting quantum interference device (SQUID), near infrared
imaging, optical fluorescence imaging, positron emission tomography
(PET), and, in highly preferred embodiments, imaging is by planar
scintigraphy or single photon emission computed tomography (SPECT).
These methods are also applicable to rapid and simple assays of
intracellular biochemical reactions in vitro and, more importantly,
as assays in instances in which presently available assay methods
are impractical or impossible, such as in vivo and in situ. For
example, in excised tissues, intracellular functions include biochemical
activities such as protein-protein binding, protein kinase activities,
protein phosphatase activities, and protease activities. Additional
examples include the activities of exo- and endo-peptidases, extracellular
metalloproteases, lysosomal proteases such as the cathepsins (cathepsin
B), as well as that of .alpha.-, .beta.-, and .gamma.-secretases,
transferases, hydrolases, isomerases, ligases, oxidoreductases,
esterases, glycosidases, phospholipases, endonucleases, ribonucleases
and .beta.-lactamases, which can be detected without the need for
tissue dispersion and growth that change the in vivo phenotype.
These methods are especially valuable for in vivo assays whereby
intracellular biological activities are detected without the need
for traumatic surgery.
By the use of the present methods, intracellular functions can
be detected in patients without the need for surgery. Accordingly,
the present invention encompasses compounds and methods for detecting
intracellular biochemical activities in living, whole animals, tissues,
or cells by administering complexes of this invention which translocate
into cells, and which are detectable in living cells at distances
removed from the cells by the presence of intervening tissue. Examples
of tissues to which the methods of the present invention can be
applied include, for example, cancer cells, in particular, central
nervous system tumors, breast cancer, liver cancer, lung, head and
neck cancer, lymphomas, leukemias, multiple myeloma, bladder cancer,
ovarian cancer, prostate cancer, renal tumors, sarcomas, colon and
other gastrointestinal cancers, metastases, and melanomas. More
specifically, the present invention can be applied to cancers such
as sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat
gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute
lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,
promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic
leukemia (chronic myelocytic (granulocytic) leukemia and chronic
lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's
disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease. The present invention
can also be used to detect the presence of enzymes associated with
diseases, conditions or disorders. Examples of diseases, conditions
or disorders to which the present invention can be applied include,
but are not limited to infection, inflammation, neurodegenerative
diseases such as Alzheimer's disease and Parkinson's disease, ALS,
hypoxia, autoimmune diseases, immune deficiencies, cardiovascular
insults such as infraction and stroke, and connective tissue disorders
such as rheumatoid arthritis, lupis and dermatomyositis, and other
specific dysfunctions of organs. Enzyme(s) associated with particular
diseases, conditions, or disorders are well known to those skilled
in the art and can be found in standard medical references, for
example, Stedman's Medical Dictionary, 26th Edition, Williams &
Wilkins, 1995, and Harrison's Principles of Internal Medicine, 14th
Edition, McGraw-Hill, 1998. The present invention therefore encompasses
peptide conjugate metal coordination complexes (and other diagnostically
useful complexes) and methods of detecting such complexes or their
reaction products in living, whole animals, tissues, or cells by
administering the present imaging complexes, especially a scintigraphic
or magnetic resonance imaging complex, which translocates into the
interior of living cells.
Kits
The present invention also provides kits comprising a quantity
of a reducing agent for reducing a preselected radionuclide, as
described, for example, by Jones et al., U.S. Pat. No. 4,452,774.
Such kits can contain a predetermined quantity of a Tat or other
cell-permeant peptide conjugate and a predetermined quantity of
a reducing agent capable of reducing a predetermined quantity of
a preselected radionuclide. Such kits can contain a predetermined
quantity of glucoheptonate. The peptide conjugate and reducing agent
can be lyophilized to facilitate storage stability. The conjugate
and reducing agent can be contained in a sealed, sterilized container.
Instructions for carrying out the necessary reactions, as well as
a reaction buffer solution(s), can also be included in the kit.
In one embodiment, the present invention provides a kit for use
in preparing cell membrane-permeant coordination complexes from
a supply of Tc-99m such as pertechnetate solution in isotonic saline
available in clinical nuclear medicine laboratories, including the
desired quantity of a selected Tat or other peptide conjugate to
react with a selected quantity of pertechnetate, and a reducing
agent such as sodium dithionite or stannous chloride in an amount
sufficient to reduce the selected quantity of pertechnetate to form
the desired peptide metal complex. In a preferred embodiment, the
kit includes a desired quantity of a selected peptide conjugate
to react with a selected quantity of reduced technetium supplied
in the kit in the form of Tc-99m-glucoheptonate, itself produced
from a stannous glucoheptonate commercial kit (Dupont Pharma), and
a reducing agent such as sodium dithionite or stannous chloride
in an amount sufficient to assure that the selected quantity of
reduced technetium produces the desired peptide metal complex.
Pharmaceutically Acceptable Salts of Peptide Complexes
Like amino acids, peptides and proteins are ampholytes, i.e., they
act as both acids and bases by virtue of the presence of various
electron-donor and acceptor moieties within the molecule. The peptide
complexes of the present invention can therefore be used in the
free acid/base form, in the form of pharmaceutically acceptable
salts, or mixtures thereof, as is known in the art. Such salts can
be formed, for example, with organic anions, organic cations, halides,
alkaline metals, etc.
The term "pharmaceutically acceptable salts" embraces
salts commonly used to form alkali metal salts and addition salts
of free acids or free bases. The nature of the salt is not critical,
provided that it is pharmaceutically acceptable. Suitable pharmaceutically
acceptable base addition salts of the present peptide complexes
include metallic salts and organic salts.
Preferred metallic salts include, but are not limited to, appropriate
alkali metal (group Ia) salts, alkaline earth metal (group IIa)
salts, and other physiologically acceptable metals. Such salts can
be prepared, for example, from aluminum, calcium, lithium, magnesium,
potassium, sodium, and zinc.
Organic salts can be prepared from tertiary amines and quaternary
ammonium salts, including in part, tromethamine, diethylamine, N,N'-dibenzyl-ethylenediamine,
chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine
(N-methyl-glucamine), and procaine.
Such salts can also be derived from inorganic or organic acids.
These salts include but are not limited to the following: acetate,
adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, camphorate, camphorsulfonate, digluconate,
cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate,
glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate,
lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate,
oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate,
mesylate, and undecanoate.
The basic nitrogen-containing groups can be quaternized with agents
such as lower alkyl halides, such as methyl, ethyl, propyl, and
butyl chloride, bromides, and iodides; dialkyl sulfates such as
dimethyl, diethyl, dibuytl, and diamyl sulfates; long chain halides
such as decyl, lauryl, myristyl, and stearyl chlorides, bromides,
and iodides; aralkyl halides such as benzyl and phenethyl bromides,
and others.
All of these salts can be prepared by conventional means from the
corresponding peptide complex disclosed herein by reacting the appropriate
acid or base therewith. Water- or oil-soluble or dispersible products
are thereby obtained as desired.
Formulations/Pharmaceutical Compositions
The compounds of the present invention can be formulated as pharmaceutical
compositions. Such compositions can be administered orally, parenterally,
by inhalation spray, rectally, intradermally, transdermally, or
topically in dosage unit formulations containing conventional nontoxic
pharmaceutically acceptable carriers, adjuvants, and vehicles as
desired. Topical administration may also involve the use of transdermal
administration such as transdermal patches or iontophoresis devices.
The term parenteral as used herein includes subcutaneous, intravenous,
intramuscular, or intrastemal injection, or infusion techniques.
Formulation of drugs is discussed in, for example, Hoover, John
E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa. (1975), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical
Dosage Forms, Marcel Decker, New York, N.Y. (1980).
Injectable preparations, for example, sterile injectable aqueous
or oleaginous suspensions, can be formulated according to the known
art using suitable dispersing or wetting agents and suspending agents.
The sterile injectable preparation may also be a sterile injectable
solution or suspension in a nontoxic parenterally acceptable diluent
or solvent, for example, as a solution in 1,3-butanediol. Among
the acceptable vehicles and solvents that may be employed are water,
Ringer's solution, and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil may be
employed, including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid are useful in the preparation of
injectables. Dimethyl acetamide, surfactants including ionic and
non-ionic detergents, and polyethylene glycols can be used. Mixtures
of solvents and wetting agents such as those discussed above are
also useful.
Suppositories for rectal administration of the compounds discussed
herein can be prepared by mixing the active agent with a suitable
non-irritating excipient such as cocoa butter, synthetic mono-,
di-, or triglycerides, fatty acids, or polyethylene glycols which
are solid at ordinary temperatures but liquid at the rectal temperature,
and which will therefore melt in the rectum and release the drug.
Solid dosage forms for oral administration may include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the compounds of this invention are ordinarily combined with one
or more adjuvants appropriate to the indicated route of administration.
If administered per os, the compounds can be admixed with lactose,
sucrose, starch powder, cellulose esters of alkanoic acids, cellulose
alkyl esters, talc, stearic acid, magnesium stearate, magnesium
oxide, sodium and calcium salts of phosphoric and sulfuric acids,
gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or
polyvinyl alcohol, and then tableted or encapsulated for convenient
administration. Such capsules or tablets can contain a controlled-release
formulation as can be provided in a dispersion of active compound
in hydroxypropylmethyl cellulose. In the case of capsules, tablets,
and pills, the dosage forms can also comprise buffering agents such
as sodium citrate, or magnesium or calcium carbonate or bicarbonate.
Tablets and pills can additionally be prepared with enteric coatings.
For therapeutic purposes, formulations for parenteral administration
can be in the form of aqueous or non-aqueous isotonic sterile injection
solutions or suspensions. These solutions and suspensions can be
prepared from sterile powders or granules having one or more of
the carriers or diluents mentioned for use in the formulations for
oral administration. The compounds can be dissolved in water, polyethylene
glycol, propylene glycol, ethanol, com oil, cottonseed oil, peanut
oil, sesame oil, benzyl alcohol, sodium chloride, and/or various
buffers. Other adjuvants and modes of administration are well and
widely known in the pharmaceutical art.
Liquid dosage forms for oral administration can include pharmaceutically
acceptable emulsions, solutions, suspensions, syrups, and elixirs
containing inert diluents commonly used in the art, such as water.
Such compositions can also comprise adjuvants, such as wetting agents,
emulsifying and suspending agents, and sweetening, flavoring, and
perfuming agents.
The amount of active ingredient that can be combined with the carrier
materials to produce a single dosage form will vary depending upon
the patient and the particular mode of administration.
Doses/Quantities of Peptide Complexes
The quantity of cell membrane-permeant peptide complex comprising
a radionuclide for use in radiolabelling and imaging, or relaxivity
metal, should be an effective amount for the intended purpose. Such
amounts can be determined empirically, and are also well known in
the art. For example, amounts of radio nuclide administered via
the present complexes can be in the range of from about 1 .mu.Ci
to about 100 mCi, preferably from about 1 mCi to about 100 mCi,
and more preferably from about 1 mCi to about 50 mCi. This amount
can be adjusted for body weight and the particular disease state,
and can be about 1 mCi/kg body weight.
For therapeutic purposes, the amount of radio nuclide administered
via the present complexes can be in the range of from about 1 mCi
to about 300 mCi, preferably from about 25 mCi to about 250 mCi,
and more preferably from about 50 mCi to about 200 mCi. Of course,
this amount can be tailored to meet the specific requirements of
the disease state being treated, and can also vary depending upon
the weight and condition of the patient as is well known in the
art. Note, for example, Clinical Nuclear Medicine, 1998, Third Edition,
Chapman & Hall Medical.
The amount of complex comprising a drug or other pharmacologically
active agent for administration to a patient to treat or prevent
a disease condition will vary with the type of drug, and will comprise
a therapeutically effective amount thereof. Drug dosages for treating
various conditions are well known in the art. Note in this regard,
for example, Goodman & Gilman's The Pharmacological Basis of
Therapeutics, 1996, Ninth Edition, McGraw-Hill, New York.
Routes of Administration
The complexes of the present invention can be administered by a
variety of methods, including, for example, orally, enterally, mucosally,
percutaneously, or parenterally. Parenteral administration is preferred,
especially by intravenous, intramuscular, subcutaneous, intracutaneous,
intraarticular, intrathecal, and intraperitoneal infusion or injection,
including continuous infusions or intermittent infusions with pumps
available to those skilled in the art. Alternatively, the complexes
can be administered by means of micro-encapsulated preparations,
for example those based on liposomes as described in European Patent
Application 0 213 523.
Treatment Regimens
The regimen for treating a patient with the compounds and/or compositions
of the present invention is selected in accordance with a variety
of factors, including the age, weight, sex, diet, and medical condition
of the patient, the severity of the condition, the route of administration,
pharmacological considerations such as the activity, efficacy, pharmacokinetic,
and toxicology profiles of the particular pharmacologically active
compounds employed.
Administration of the drug complexes disclosed herein should generally
be continued over a period of several days, weeks, months, or years.
Patients undergoing treatment with the drug complexes disclosed
herein can be routinely monitored to determine the effectiveness
of therapy for the particular disease or condition in question.
Continuous analysis of the data obtained by these methods permits
modification of the treatment regimen during therapy so that optimal
amounts of the pharmacologically active substance in the peptide
complex are administered, and so that the duration of treatment
can be determined as well. Thus, the treatment regimen/dosing schedule
can be rationally modified over the course of therapy so that the
lowest amounts of drug compound is administered, and so that administration
of such compounds is continued only so long as is necessary to successfully
treat the disease or condition.
Monitoring Devices/Procedures
Detection methods useful in practicing the present invention include,
but are not limited to magnetic resonance, superconducting quantum
interference device (squid), optical imaging (e.g. fluorescence
tomography, NIRF imaging systems, in vivo bioluminescence, and endoscopic
fluorescence), positron emission tomography, and in particular,
planar scintigraphy or single photon emission computed tomography
(SPECT). Alternative methods of detection include gamma counting,
scintillation counting, scanning radiograms, densitometry and fluorography.
These detection methods can be employed during or after an effective
time interval for diagnosis or imaging subsequent to administering
a peptide complex of the present invention. Such effective time
intervals are well known in the art, or can be determined by routine
experimentation employing methods such as those disclosed herein.
Although the examples hereinafter provided contain many specificities,
these should not be construed as limiting the scope of the invention,
but as merely providing illustrations of some of the aspects of
the present invention.
EXAMPLE 1
Preparation of acetyl-GRKKRRQRRR-AHA-.epsilon.KGC-amide trifluoroacetate
A Tat peptide (residues 48-57, GRKKRRQRRR (SEQ ID NO: 7)) conjugate
was prepared by solid phase peptide synthesis using N-.alpha.-FMOC-protected
amino acids and standard BOP/HOBt coupling chemistry (Merifield
et al., Biochemistry 21:5020-5031, 1982; Houghten, Proc Natl Acad
Sci USA 82:5131-5135, 1985; Lin, et al., Biochemistry 27:5640-5645,
1988), except for the .epsilon.-Lys residue, which used an N-.alpha.-tBOC,
N-.epsilon.-FMOC-Lys residue to generate the desired peptide-based
N.sub.3S chelating group for an incoming metal (Lister-James, et
al., Q J Nucl Med 41:111-118, 1997). AHA represents aminohexanoic
acid as an example of a non-functional linker between the Tat 48-57
residues and the chelating moiety. The peptide was amino acetylated,
carboxy amidated, and deprotected by standard methods (Merifield
et al., Biochemistry 21:5020-5031, 1982; Houghten, Proc Natl Acad
Sci USA 82:5131-5135, 1985; Lin, et al., Biochemistry 27:5640-5645,
1988). The peptide was purified (>94%) by preparative C.sub.18
reversed-phase HPLC using as eluent 0.1% trifluoroacetic acid in
water (0.1% TFA/H.sub.2O) modified with 0.1% trifluoroacetic acid
in 90% acetonitrile/10% water (0.1% TFA/(90% CH.sub.3CN/H.sub.2O))
by a linear gradient (0% to 60% over 60 min) (peptide R.sub.t=21
min). The identity of the peptide conjugate was confirmed by amino
acid analysis (13 proteinogenic amino acids: Glu 1; Gly 2; Cys 1;
Lys 3, Arg 6) and electrospray mass spectrometry (m/z: 1839.0; calc:
C.sub.74H.sub.143N.sub.37O.sub.16S.sub.1, 1839.27). The sequence
was confirmed as acetyl-GRKKRRQRRR-AHA-.epsilon.KGC-amide ((SEQ
ID NO: 30).
EXAMPLE 2
Preparation of Radiolabeled acetyl-GRKKRRQRRR-AHA-.epsilon.KGC-amide(Tc.sup.v-99m)
trifluoroacetate
The Tat peptide conjugate complex of Example 1 was labeled with
Tc-99m by ligand exchange using Tc-99m-glucoheptonate as the ligand
exchange reagent (Lister-James et al., J. Nucl. Med. 38:105-111,
1997). A commercially available stannous glucoheptonate radiopharmaceutical
kit (Glucoscan, DuPont Pharma, Billerica, Mass.) was reconstituted
with 1.0 ml of (Tc-99m)sodium pertechnetate (50 mCi) in isotonic
saline obtained by eluting a commercial radionuclide Mo-99/Tc-99m
generator, and allowed to stand for 15 min at room temperature.
In a small glass vial, Tat peptide conjugate (1 mg) was dissolved
in 0.9% saline (1 ml). Then, (Tc-99m)glucoheptonate (250 .mu.l)
was added and the reaction allowed to proceed at room temperature
for 15 min. Radiochemical yield (>95%) of the oxotechnetium complex
(FIG. 2) and purity (.gtoreq.90%) were determined by silica gel
TLC using 15% TFA and radiometric detection (Bioscan)((Tc-99m)-peptide
complex, R.sub.f 0.24; (Tc-99m)-glucoheptonate, R.sub.f 0.95; (Tc-99m)-TcO.sub.4.sup.-,
R.sub.f 0.95).
EXAMPLE 3
Preparation of acetyl-GRKKRRQRRR-AHA-.epsilon.KGC-amide-fluorescein-maleimide
trifluoroacetate
The Tat peptide conjugate of Example 1 was labeled with fluorescein
according to Vives et al. (J. Biol. Chem., 272:16010-16017, 1997).
In a small glass vial, Tat peptide conjugate (1 mg) was dissolved
in phosphate buffered saline (pH 7.4) and reacted with 1.2 eq of
fluorescein maleimide dissolved in dimethylformamide for 2 hours
in the dark at room temperature. The reaction was monitored by RP-HPLC
at both 211 nm and 440 nm. Fluorescent peptides were purified by
HPLC (purity >97%) using the above gradient conditions and lyophilized
in the dark. The identity of the desired fluorescein labeled peptide
was confirmed by electrospray mass spectrometry (m/z: 2211.0).
EXAMPLE 4
Solutions for Cell Uptake Experiments
Control solution for cell uptake experiments was a modified Earle's
balanced salt solution (MEBSS) containing (mM): 145 Na.sup.+, 5.4
K.sup.+, 1.2 Ca.sup.2+, 0.8 Mg.sup.2+, 152 Cl.sup.-, 0.8H.sub.2PO.sub.4.sup.-,
0.8 SO.sub.4.sup.-, 5.6 dextrose, 4.0 HEPES, and 1% bovine calf
serum (vol/vol), pH 7.4.+-.0.05. A 130 mM K.sup.+/20 mM Cl.sup.-
solution was made by equimolar substitution of potassium methanesulfonate
for NaCl as described by Piwnica-Worms et al. (J. Gen. Physiol.,
81:731-748, 1983).
EXAMPLE 5
Cell Culture
Monolayers of human epidermoid carcinoma KB 3-1 cells and the colchicine-selected
KB 8-5 and KB 8-5-11 derivative cell lines were grown as previously
described (Akiyama et al., Somatic Cell Mol. Genet., 11:117-126,
1985; Piwnica-Worms et al., Cancer Res., 53:977-984, 1993). Briefly,
cells were plated in 100-mm Petri dishes containing seven 25-mm
glass coverslips on the bottom and grown to confluence in DMEM (GIBCO,
Grand Island, N.Y.) supplemented with L-glutamine (1%), penicillin/streptomycin
(0.1%), and heat-inactivated fetal calf serum (10%) in the presence
of 0, 10 and 100 ng/ml colchicine, respectively. Human Jurkat leukemia
cells and Hela tumor cell lines were maintained in RPMI supplemented
with 5-10% fetal calf serum, penicillin, streptomycin, and L-glutamine
at 37.degree. C. in an atmosphere of 5% CO.sub.2 (Peng et al., Science,
277:1501-1505,1997).
EXAMPLE 6
Cell Accumulation and Washout Studies of Tat-Peptide Conjugate
Metal Complexes
Coverslips with confluent cells were used for studies of cell transport
and kinetics of labeled Tat peptide conjugate complexes as previously
described (Piwnica-Worms et al., Cancer Res., 53:977-984, 1993).
Cells were removed from culture media and pre-equilibrated for 15-30
seconds in control buffer. Accumulation experiments were initiated
by immersing coverslips in 60-mm glass Pyrex dishes containing 4
ml of loading solution consisting of MEBSS with 7 nM to 8 .mu.M
of the peptide conjugate of Example 2 (1-2 .mu.Ci/ml). Coverslips
with cells were removed at various times, rinsed three times in
25 ml ice-cold isotope-free solution for 8 seconds each to clear
extracellular spaces, and placed in 35-mm plastic Petri dishes.
Cells were extracted in 1% sodium dodecylsulfate with 10 mM sodium
borate before protein assay by the method of Lowry (Lowry et al.
J. Biol. Chem., 193:265-275, 1951) (KB cells) or by BCA analysis
(pierce Chemical Co.) using bovine serum albumin as the protein
standard. Aliquots of the loading buffer and stock solutions also
were obtained for standardizing cellular data with extracellular
concentration of each Tc-complex. Cell extracts, stock solutions,
and extracellular buffer samples were assayed for gamma activity
in a well-type sodium iodide gamma counter (Cobra II, Beckman).
The absolute concentration of total Tc-complex in solution was determined
from the peptide stock solutions and specific activity of technetium,
based on equations of Mo/Tc generator equilibrium (Lamson et al.,
J. Nucl. Med., 16:639-641, 1975).
Characterization of accumulation of Tc-99m-peptide complex was
also performed for nonadherent cell lines such as human Jurkat leukemia
cells with minor modifications of methods described in the literature
(Bosch et al., Leukemia, 11: 1131-1137, 1997). Transport experiments
were performed in siliconized microfuge tubes and initiated by addition
of 732.5 .mu.l of cells at 2-3.times.10.sup.6 cells/ml to 10 .mu.l
of buffer containing Tc-99m-peptide complex and 7.5 .mu.l of vehicle
alone or of any added drug in vehicle at 100-fold the desired concentration.
The tubes were incubated in a 37.degree. C. water bath with occasional
mixing. The reaction was terminated by centrifuging 250 .mu.l aliquots
from the reaction for 10 seconds through 800 .mu.l of a 75:25 mixture
of silicon oil, density=1.050 (Aldrich) and mineral oil, density=0.875
(Acros). An aliquot of the aqueous phase was obtained to normalize
extracellular concentration of the complex to cell-associated activity,
then the oil and aqueous phases were aspirated and the cell pellet
extracted in 0.5 ml of 1% SDS, 10 mM sodium borate. For tracer washout
experiments, cells were first incubated to plateau uptake (10 min)
in loading buffer (37.degree. C.), collected by rapid centrifugation
and the pellet resuspended in 50 ml MEBSS (4.degree. C.) to clear
extracellular tracer. Following another rapid spin, the cell pellet
was resuspended in isotope-free MEBSS (37.degree. C.) and the experiment
terminated as above after various times in warm washout buffer.
Radioactivity of the cell pellet, buffers and stocks were determined
on a gamma counter (Cobra II, 130-165 keV window) and cell protein
was determined by the BCA assay (Pierce). Transport data are reported
as fmol Tc-complex (mg protein).sup.-1 (nM.sub.0).sup.-1 as previously
described, with (nM.sub.0).sup.-1 representing total concentration
of peptide conjugate in the extracellular buffer (Piwnica-Worms
et al., Circulation, 82: 1826-1838, 1990).
When exposed to radioactive Tc-99m-Tat peptide metal complex, human
Jurkat leukemia cells rapidly accumulated the complex, approaching
a plateau within 2 minutes (FIG. 3). Steady-state values for the
Tc-99m-Tat peptide metal complex in Jurkat cells was 116.+-.3 fmol
(mg protein).sup.-1 (nM.sub.0).sup.-1 (n=4). Given a typical cell
water space of 4 .mu.l (mg protein).sup.-1, this would indicate
an in/out ratio for the complex of .about.30, directly demonstrating
that the complex is rapidly and highly concentrated within cells.
When continuously exposed to the complex, cells were observed to
maintain this plateau for at least 1 hour.
To further characterize transport of the Tc-99m-Tat peptide metal
complex, plateau accumulation of the agent in Jurkat cells after
10 minutes of incubation was determined as a function of extracellular
concentration of the radiopharmaceutical. While readily detectable
at concentrations as low as 7 nM, cell content of the Tat-complex
showed evidence of concentration-saturation as extracellular concentrations
rose into the range of 8 .mu.M (FIG. 4). Curve fitting of the data
suggested half-maximal accumulation of the complex occurred at .about.3
.mu.M.
To further define the interactions of the complexes with-cells,
Jurkat cells were incubated with Tc-99m-complexes in MEBSS buffer
alone or buffer containing 130 mM K.sup.+/20 mM Cl.sup.- and 1 .mu.g/ml
of the potassium ionophore valinomycin. Under these conditions,
electrical potentials of the mitochondrial membrane (.DELTA..PSI.)
and plasma membrane (E.sub.m) are depolarized toward zero, eliminating
the inward driving force for uptake of hydrophobic cationic or amphipathic
molecules (Piwnica-Worms et al., Circulation, 82:1826-1838,1990).
However, while the complex might be characterized as amphipathic,
net uptake of the complex under isoelectric conditions was not decreased
compared to control buffer, suggesting that the mechanism of uptake
was independent of membrane potential (data not shown).
Because several membrane permeant peptides have been reported to
be accumulated within cells by mechanisms related to cytoskeletal
function (Elliot and O'Hare, Cell, 88:223-233, 1997), several inhibitors
known to impact microtubulin, actin microfilament and various cytoskeletal-mediated
vesicular transport pathways were tested in Jurkat cell assays.
Colchicine (100 ng/ml), taxol (1 .mu.M), nocodozole (5 .mu.g/ml),
cytochalasin D (1 .mu.M), brefeldin A (2.5 .mu.g/ml) and wortmannin
(100 nM) each had no significant effect on net cell uptake of this
Tat-peptide metal complex, indicating that the pathway for accumulation
of this agent is by a previously uncharacterized mechanism (data
not shown). Furthermore, ice-cold buffer (4.degree. C.) only modestly
inhibited net accumulation of the complex, further pointing to a
unique cell membrane translocation pathway not highly dependent
on cellular metabolism. Cellular washout of the non-functional peptide
complex of Example 2 which had been previously preloaded into Jurkat
cells also showed very rapid kinetics. Washout was .about.90% complete
within 20 minutes (FIG. 5). This demonstrates that the majority
of non-functionalized Tat peptide conjugate is not retained within
cells when extracellular concentrations of the peptide are lowered.
Only a residual level of peptide representing <10% of peak activity
remained in a slowly exchanging or retaining compartment.
EXAMPLE 7
Fluorescence Microscopy
Exponentially growing human KB-8-5 epidermoid carcinoma cells on
coverslips were rinsed in serum-free MEBSS (37.degree. C.) followed
by incubation in serum-free MEBSS containing the fluorescein labeled
Tat-peptide conjugate (1 .mu.M) at 37.degree. C. for 15 min. Subsequently,
cells on covers lips were fixed in 4% (v/v) formaldehyde in PBS
at room temperature and then rinsed 3 times with PBS (1 min each).
Cells were then stained and mounted with anti-fading mounting medium
containing propidium iodide (1 .mu.g/ml) following the recommended
procedures of the manufacturer (Vectashield). The distribution of
the fluorescence was analyzed on a Zeiss confocal laser fluorescence
microscope equipped with a mercury lamp, oil immersion objectives
and a CCD interfaced to a PC. Propidium iodide distribution was
interrogated using 340-380 nm excitation and 430 nm emission, while
fluorescein distribution was interrogated using 450-490 nm excitation
and 520 nm emission.
To localize the subcellular distribution of the Tat-peptide conjugate,
uptake experiments were performed with the fluorescein derivatized
conjugate using human KB-3-1 and KB-8-5 epidermoid carcinoma cells.
Confocal microscopy revealed rapid cytoplasmic and nuclear accumulation
of the fluorescein derivatized conjugate at 0.5 .mu.M extracellular
concentration of the agent. Both KB-3-1 cells (FIG. 6) and KB-8-5
cells (not shown) displayed a similar pattern and intensity of staining.
Overall, the nuclear staining pattern of most fluorescent cells
was suggestive of cytosolic and nucleolar localization of the peptide
conjugate (FIG. 6).
EXAMPLE 8
Preparation of Caspase-3-Cleavable Metal and Fluorescein Conjugates
Caspase-3 cleavable Tat peptide conjugate was prepared by solid
phase peptide synthesis using N-.alpha.-FMOC-protected amino acids
and standard BOP/HOBt coupling chemistry as in Example 1. The peptide
made incorporated a known caspase-3 cleavable sequence (DEVD) between
the Tat peptide and the chelate. As described previously in Example
1, the peptide was amino acetylated, carboxy amidated and deprotected
by standard methods. The peptide was purified (>94%) by preparative
C.sub.18 reversed-phase HPLC (see Example 1), and the identity of
the peptide conjugate was confirmed by amino acid analysis and electrospray
mass spectrometry (m/z: 2412.23; calc: C.sub.96H.sub.175N.sub.43O.sub.18S.sub.1,
2411.79). The sequence was confirmed as acetyl-GRKKRRQRRR-GDEVDG-.epsilon.KGC-amide
(SEQ ID NO: 31).
The caspase-3 cleavable Tat peptide conjugate was labeled with
Tc-99m by ligand exchange using Tc-99m-glucoheptonate as the ligand
exchange reagent as described in Example 2. Radiochemical yield
(>95%) of the oxotechnetium and purity (>90%) were determined
by silica gel TLC using 15% TFA and radiometric detection (Bioscan).
The (Tc-99m)-peptide complex showed an R.sub.f=0.33, readily distinguished
from (Tc-99m)-glucoheptonate (R.sub.f=0.95) and (Tc-99m)-TcO.sub.4.sup.-
(R.sub.f=0.95).
The caspase-3 cleavable Tat peptide was also readily complexed
with Re by ligand exchange (Lister-James et al., J. Nucl. Med. 38:105-111,
1997). To 0.1 ml of a freshly prepared solution of glucoheptonate
and reducing agent (200 mg (0.81 mmol) sodium .alpha.-D-glucoheptonate
and 18.4 mg (0.082 mmol) tin (II) chloride dihydrate in 1 ml distilled
water) was added 0.1 ml of a solution of ammonium perrhenate (14.9
mg (0.055 mmol) in 1 ml) and the mixture allowed to stand for 15
min at room temperature. To the mixture was added 1 mg of Tat peptide
caspase-3 cleavable conjugate and the reaction allowed to proceed
at room temperature for 30 minutes. The conjugate was purified by
RP-HPLC as in Example 1. The identity of the ReO peptide conjugate
was confirmed by electrospray mass spectrometry (m/z: 2612.0; calc:
C.sub.96H.sub.172N.sub.43O.sub.19S.sub.1Re.sub.1, 2611.73).
RP-HPLC analysis using the same solvent gradient system and radiometric
detection as previously described in Example 1 revealed two closely
eluting peaks for the Tc-99m complex (R.sub.t,1=23.9 min; R.sub.t,2=25.8
min). RP-HPLC analysis and UV detection revealed two corresponding
peaks for the Re complex (R.sub.t,1=21.3 min; R.sub.t,2=25.8 min),
again consistent with formation of the expected isomers of the oxometal
complexes.
The caspase-3 cleavable Tat peptide conjugate was also labeled
at the C-terminal thiol of the peptide chelator with fluorescein
maleimide using the same procedure as described in Example 3. The
reaction was monitored by RP-HPLC at both 211 nm and 440 nm. The
fluorescent peptide was purified by RP-HPLC (R.sub.t=33.5 min; purity
>97%) using the gradient conditions given in Example 3, and lyophilized
in the dark. The identity of the desired fluorescein labeled peptide
was confirmed by electrospray mass spectrometry (m/z: 2840.0).
EXAMPLE 9
Cleavage of the Caspase-3 Cleavable Linker In Vitro and In Situ
In small reaction vials, Tat peptide chelate as the fluorescein
tagged conjugate of Example 8 was incubated with and without recombinant
human active caspase-3 in commercially available reaction buffer
(caspase buffer, Invitrogen). In vial 1 was peptide conjugate in
buffer without caspase-3; in vial 2 was peptide conjugate with active
caspase-3; and in vial 3 was stock peptide conjugate. After 6 hrs
of incubation to assure completion of the reaction, the reaction
mixtures were spotted at the origin of silica gel TLC plates, developed
in 15% TFA, and analyzed under an UV lamp. While the unreacted peptide
chelate stock and peptide chelate incubated in buffer alone retained
an R.sub.f=0.33, peptide chelate incubated in the presence of caspase-3
resulted in disappearance of the R.sub.f=0.33 species and appearance
of a peptide cleavage product with R.sub.f=0.66. These data are
consistent with cleavage of the Tat peptide conjugate at the D-G
cleavage site, thereby releasing the small molecular weight C-terminus
G-.epsilon.KGC-fluorescein fragment identified near the solvent
front on TLC. This represents direct evidence for successful synthesis
of a caspase-3-cleavable Tat peptide imaging conjugate.
Human Jurkat leukemia cells express pro-caspase-3. Apoptosis can
be induced by pre-incubation of Jurkat cells for 5 hr in medium
containing C6-ceramide, a permeant phospholipid known to activate
the cell death program (Herr, et al., EMBO J. 16:6200-6208, 1997;
Jayadev S, et al., J Biol Chem 270:2047-2052, 1995). After pre-incubation
of Jurkat cells in MEBSS buffer at 370.degree. C. in the absence
(untreated) or presence of 5 .mu.M C6-ceramide, 1 .mu.M of the caspase-3
cleavable fluorescein tagged Tat peptide of Example 8 was added
to the MEBSS buffer for 30 minutes. Untreated and apoptotic cells
were then spun through oil (see Example 6) to clear extracellular
spaces of Tat peptide, and the intact cells in the pellet were allowed
to incubate for 5 minutes at 37.degree. C. The oil was quickly suctioned
off, the reaction terminated with cell lysis buffer (1% SDS, 10
mM sodium borate), and the cell extract centrifuged (500.times.g
for 10 min) to pellet debris and precipitates. The supernatant was
removed, lyophilized overnight, and resuspended in 500 .mu.l of
water. In untreated cell lysates, RP-HPLC analysis at 440 nm to
observe fluorescein (see Example 3) showed the presence of a peak
at R.sub.t=33.5 min, consistent with parental Tat peptide conjugate
(FIG. 7). In C6-ceramide-treated cells, however, no such species
was observable (FIG. 7). These results demonstrate the rapid cleavage
of the Tat-peptide conjugate comprising a caspase-3-reactive linker
moiety in living cells upon activation of caspase-3.
The above experiment was repeated using the Tc-99m-Tat peptide
of Example 8. Cells were treated as above except that the Tc-99m-Tat
peptide was used, and there was no washout or post-incubation period.
Tc99-m and protein content were determined using published methods
(Bosch et al., Leukemia 11:1131-37, 1997). Cells induced to undergo
apoptosis by treatment with C6-ceramide showed enhance uptake of
Tc-99m, again showing that the presence of the caspase-3 cleavable
linker resulted in identification of apoptotic cells.
EXAMPLE 10
Imaging Studies
FVB mice were anesthetized with metophane anesthesia. Tc-99m-Tat-peptide
complex of Example 8 (125 .mu.Ci in 50 .mu.l saline) was injected
via a tail vein into mice positioned under a gamma scintillation
camera (Siemens Basicam, Siemens Medical Systems, Iselin, N.J.;
5 mm pinhole collimator; 20% energy window centered over 140 keV
photopeak of Tc-99m). Sequential posterior images of mice were collected
at one frame/minute for 60 min with a 128.times.128 matrix and corrected
for radioactive decay using a PC platform and standard commercial
image analysis software. Accumulation, of Tc-99m-Tat-peptide complex
was analyzed by manually drawing regions-of-interest over various
organs and subtracting background radioactivity determined from
a region-of-interest placed adjacent to the thorax of each mouse.
No corrections were made for scatter or attenuation. Whole body
distribution of the complexes are presented in pseudo gray scale
images with or without a saturation cutoff filter to highlight contrast
differences in various organs.
The Tc-99m-Tat peptide initially showed a whole body microvascular
distribution, followed by rapid and abundant renal localization
and excretion. By 30 minutes post injection of the imaging agent,
the only site of imagable radioactivity was the urinary bladder
(FIG. 8). There was a remarkable absence of liver activity or other
background activity that would potentially interfere with the imaging
of specific organ tissues or tumors. This rapid distribution pattern
is consistent with the in vitro cell kinetic and localization data,
but the rapidity of the renal excretion was unexpected.
Next, direct demonstration of the feasibility of imaging caspase-3
activity in vivo in a living organism using gamma scintigraphy is
shown. Massive hepatic apoptosis can be induced within 1-2 hours
in mice following the intravenous injection of anti-Fas antibody
(Ogasawara, et al., Nature 364:806-809; 1993; Blankenberg, et al.,
Proc Natl Acad Sci USA 95:6349-6354, 1998). The Fas receptor is
expressed on liver, kidney, thymus, gonads and subsets of leukocytes
(Ogasawara, et al., Nature 364:806-809; 1993). Thus, to test the
specific localization of the caspase-3-cleavable Tc-99m-Tat peptide
agent of Example 8 in organs undergoing apoptosis in vivo, a published
procedure was used to image mice following the induction of apoptosis
(Blankenberg, et al., Proc Natl Acad Sci USA 95:6349-6354, 1998).
FVB mice were administered purified hamster anti-Fas mAb by i.v.
injection and allowed to recover for 45 minutes prior to imaging.
Following metofane anesthesia, 200 .mu.Ci of Tc-99m-Tat chelate
was administered by tail vein injection, and mice were immediately
positioned for imaging on a gamma scintillation camera. In untreated
mice, the Tc-99m-Tat peptide initially showed a whole body distribution,
followed by rapid and abundant renal localization and excretion,
as expected. In contrast, mice pre-treated with anti-Fas mAb showed
abundant hepatic and renal retention of radioactivity 30 minutes
post injection, consistent with caspase-3-induced cleavage and retention
of the imaging fragment within the target organs (FIG. 9, right).
These images represent the first example of imaging caspase-3 activity
in vivo, and demonstrate the utility of this approach in imaging
with cell membrane-permeant peptide conjugates.
EXAMPLE 11
Preparation of D-Amino Acid Containing Peptide Conjugates
Peptide conjugates were prepared by solid state peptide synthesis
as described in Example 1 using D N-.alpha.-FMOC-protected amino
acids and standard BOP/HOBt coupling chemistry, except for the .epsilon.-Lys
residue which used an N-.alpha.-tBOC, N-.epsilon.-FMOC-Lys to direct
peptide coupling to the .epsilon.-amine. Some peptides were either
N-terminus acetylated or biotinylated, and all peptides were C-terminus
amidated and deprotected by standard methods. Peptides were purified
by C.sub.18 reversed-phase HPLC as described in Example 1. A single
HPLC peak was observed for each peptide conjugate. The identity
of the peptide conjugates was confirmed by amino acid analysis and
electrospray mass spectrometry.
The following peptide conjugates were synthesized and characterized.
The stereoisomeric identity of the membrane permeant peptide (Tat
basic) domains and the chelation domains (.epsilon.KGC) are indicated
for each group. AHA represents aminohexanoic acid, an amino acid
residue lacking a chiral center used in this example as a non-functional
linker between the membrane permeant peptide and the metal chelation
domains.
TABLE-US-00001 L L Acetyl-GRKKRRQRRR-AHA-.epsilon.KGC-amide Conjugate
1 SEQ ID NO: 30 Acetyl-RKKRRQRRR-AHA-.epsilon.KGC-amide Conjugate
2 SEQ ID NO: 32 Biotin-RKKRRQRRR-AHA-.epsilon.KGC-amide Conjugate
3 SEQ ID NO: 32 L D Acetyl-GRKKRRQRRR-AHA-.epsilon.KGC-amide Conjugate
4 Acetyl-RKKRRQRRR-AHA-.epsilon.KGC-amide Conjugate 5 NH.sub.2-GRKKRRQRRR-AHA-.epsilon.KGC-amide
Conjugate 6 NH.sub.2-RKKRRQRRR-AHA-.epsilon.KGC-amide Conjugate
7 D D Acetyl-GRKKRRQRRR-AHA-.epsilon.KGC-amide Conjugate 8 Acetyl-RKKRRQRRR-AHA-.epsilon.KGC-amide
Conjugate 9 NH.sub.2-GRKKRRQRRR-AHA-.epsilon.KGC-amide Conjugate
10 NH.sub.2-RKKRRQRRR-AHA-.epsilon.KGC-amide Conjugate 11 D L Acetyl-RKKRRQRRR-AHA-.epsilon.KGC-amide
Conjugate 12 Biotin-RKKRRQRRR-AHA-.epsilon.KGC-amide Conjugate 13
D D Biotin-RAARRAARR-AHA-.epsilon.KGC-amide Conjugate 14
The conjugates identified in Table 2 were prepared by solid-phase
peptide synthesis using L- or D-N-.alpha.-FMOC-protected amino acids
as indicated and standard BOP/HOBt coupling chemistry as is known
in the art, with the exception that N-.epsilon.-FMOC-protected Lys
(*K) was used in the chelation sequence to direct orthogonal peptide
coupling and free the .alpha.-amino for coordination with the incoming
metal. Peptides were purified (>94%) by preparative C.sub.18
reverse-phase HPLC, and single HPLC peaks were observed for each
peptide conjugate. The identity of all peptides was confirmed by
amino acid analysis and electrospray mass spectrometry as is known
in the art.
EXAMPLE 12
Preparation of [99 mTc.sup.vO Tat-Peptide Trifluroracetate
The Tat-peptide conjugates prepared in Example 11 were labeled
with .sup.99mTc by ligand exchange using [.sup.99mTc] glucoheptonate
as described in Example 2.
EXAMPLE 13
Preparation of [Re.sup.vO] Tat-Peptide Trifluoroacetate
The Tat-peptide conjugates prepared in Example 11 were also reacted
with Re by ligand exchange using [Re]glucohemtoanate as the ligand
exchange reagent by the method used in Example 2. To 0.1 ml of a
freshly prepared solution of 0.81 mmol sodium .alpha.-D-glucoheptonate
and 0.082 mmol tin(II) chloride dihydrate was added 0.1 ml of a
solution of 0.055 mmol ammonium perrhenate and the mixture allowed
to stand for 15 minutes at room temperature. To the mixture was
added 1 mg of the Tat-peptide conjugate (0.41 .mu.mol) in water
and the reaction allowed to proceed at room temperature for 30 minutes.
Reversed phase HPLC analysis was performed as previously described
and the desired fractions collected. The identity of the isolated
[Re]Tat-peptide complexes was confirmed by electrospray mass spectrometry.
EXAMPLE 14
Cellular Uptake and Washout Studies of [.sup.99mTc] D-Tat-Peptide
Conjugates
Control solution for the cellular uptake experiments was the modified
Earle's balanced salt solution (MEBSS) described in Example 4.
Kinetic experiments of [.sup.99mTc]D-Tat-peptide complexes were
performed in Jurkat leukemia cells suspended in MEBSS with minor
modification of the methods described in Example 6. Transport experiments
were performed in siliconized microfuge tubes and initiated by addition
of 732.5 .mu.l of cells at 2-3.times.10.sup.6 cells/ml to 10 .mu.l
of MEBSS containing [.sup.99mTc]D-Tat-peptide complex and 7.5 .mu.l
of vehicle alone or of any added drug in vehicle at 100 fold the
desired concentration. Unless stated otherwise, [.sup.99mTc]D-Tat-peptide
complex was added to MEBSS accompanied by a molar excess of unlabeled
D-Tat-peptide as obtained directly from the labeling procedure.
The final total peptide concentration was 7 nM to 8 .mu.M (1-2 .mu.Ci/ml).
The tube were incubated at 37.degree. C. and the reaction terminated
as previously described. For peptide washout experiments, cells
were first incubated to plateau uptake (20 minutes) in MEBSS loading
buffer at 37.degree. C., collected by rapid centrifugation and the
pellet resuspended in 50 ml of isotope-free MEBSS at 4.degree. C.
to clear extracellular tracer. Following another rapid spin, the
cell pellet was resuspended in isotope-free MEBSS at 37.degree.
C. for various times and the reaction terminated as described previously.
Protein assays and determination of gamma activity were as described
in Example 6. Absolute concentration of total [Tc]Tat-peptide complex
in solution was determined from the specific activity of Tc, based
on equations of Mo/Tc generator equilibrium (Lamson et al., J. Nucl.
Med., 16:639-641, 1975). Transport data are reported as pmol of
peptide.sub.i(mg protein).sup.-1 (.mu.M.sub.0).sup.-1, wherein peptide.sub.i
represents total peptide conjugate within the cells and (nM.sub.0).sup.-1
represents concentration of total peptide conjugate in the extracellular
buffer.
As shown in Table 1, stereoisomeric substitution of D amino acids
in the metal chelation motif resulted in no significant change in
overall accumulation levels in Jurkat cells. Neither deletion of
the N-terminus Gly (Conjugates 5 and 7) nor deletion of the N-terminus
acetyl (Conjugates 6 and 7) conferred any significant differences
in overall cell penetration.
Conversely, peptide conjugates synthesized by solid phase methods
with all D-amino acids comprising both the -.epsilon.KGC chelation
motif and the membrane permeant domain (Conjugates 8-11), showed
an 8 to 9-fold increased accumulation. Again, neither deletion of
the N-terminus Gly (Conjugates 9 and 11) nor deletion of the N-terminus
acetyl (Conjugates 10 and 11) conferred any significant differences
in the overall enhanced levels of cell penetration.
Direct proof that this stereospecific enhancement of membrane penetration
was conferred by the membrane permeant domain was obtained by synthesis
of mixed peptides where natural L-amino acids comprised the -.epsilon.KGC
chelation motif and D-peptides comprised the membrane permeant domain
(Conjugates 12 and 13). These mixed peptides also showed 8- to 9-fold
increased accumulation in Jurkat cells (Table 1). Neither deletion
of the N-terminus Gly (Conjugate 12) nor substitution of the N-terminus
acetyl with biotin (Conjugate 13) conferred any significant differences
in the overall enhanced levels of cell penetration. There was a
minor trend for the D peptides to show slightly greater residual
activity remaining within the cell 30 minutes after a wash in isotope-free
buffer (Table 1). However, the net gain in cell uptake conferred
by the D peptide permeation motif far exceeded this slight increase
in residual binding as shown by the enhanced uptake/washout (U/W)
ratios for the D peptides. Further demonstration of the importance
of the specific D sequences identified by these experiments is shown
by comparison to another highly basic all D peptide (Conjugate 14).
This all D peptide was no different than the native Tat peptide
chelate (Conjugate 1) in overall cell uptake (Table 1). Direct comparative
data of cell uptakes of peptide conjugates comprising various combination
of stereoisomers of the permeation/chelation motifs (L/L; L/D; D/D;
and D/L) are shown in FIG. 10. These data reinforce the large and
unanticipated enhancement of cellular accumulation of [.sup.99mTc]Tat-peptide
complexes conferred by the use of D-amino acids for synthesis of
the membrane permeant domain.
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