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METHODS AND COMPOSITIONS FOR TREATING CANCER
This worle was supported, in part, by National Institutes of Health (NIH)
Grant
No. ES-09996 and No.l P20 RR-17659. The LTS Government has an interest in this
invention.
BACKGROUND OF THE INVENTION
The exposure of humans and other mammals to heavy metals ranges can
generate useful, therapeutic anti-cancer or anti-inflammatory responses to
severe toxic
and poisoning reactions (Suzulci-Kurasaki et al 1997 J. Histochem. CytochenZ,
45:1493-1501). Unfortunately, the differences in heavy metal concentration
that may
result in a therapeutic reaction versus an adverse reaction are very small.
The
mammalian protein, metallothionein is produced by several tissues and
circulates
throughout the bloodstream. It mediates renal exposure and toxicity to heavy
metals
by complexing with the heavy metals in vivo. Metals that complex with
metallothionein include metals encountered during environmental and
occupational
exposure, such as cadmium and mercury; metals involved in genetic disorders,
e.g.,
copper; as well as metals used for therapeutic purposes, e.g., gold and
platinum.
Thus, metallothionein metabolism is critical in the fields of human physiology
and
therapeutics.
For example, environmental and occupational exposure to cadmium is
widespread, but mostly chronic and low-level. Whether ingested or inhaled, the
majority of absorbed cadmium eventually complexes with metallothionein
(Nordberg,
M. 1984 Envirorr.. Health Perspect., 5:13-20; Chan, HM et al, 1993 Toxicol.
Appl.
Pharacol., I?3:89-96). The resulting Cd metallothionein complex is small
enough (-7
IcDa) to be freely filtered through the renal glomerulus into the proximal
tubular fluid
before reuptalce into proximal tubular cells (Foullees, EC, "Role of
naetallothionein in
epithelial tr~a~rsport and sequestration of cadmium" in Metallothionein in
Biology and
Medicine, Klassen CD, Suzulci KT (eds), RC Press, Boca Raton, FL, 1991:pp171-
182). Although neither the metallothionein apoprotein nor the zinc complex
appears
toxic, the heavy metal complex Cd-metallothionein is a renal tubular toxin.
Damage
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WO 2005/072270 PCT/US2005/002023
caused by such a toxin includes proteinuria, glucosuria, and aminoaciduria, or
in more
severe cases, acute tubular necrosis or chronic renal failure (Rehm S, Waalkes
MP,
1990 Toxicol. Appl. Plaarmacol. 104:94-105).
Heavy metals when used therapeutically are also limited by their renal
toxicity.
Metallothionein carries the commonly prescribed therapeutic heavy metals, gold
and
cisplatinum, throughout the body. Gold therapy is predominantly used to treat
rheumatoid arthritis, but the duration and dosage of gold administration are
limited by
nephrotoxicity (Saito, S and I~urasaki, M., 1996 Res. Cof~xrnun. Mol. Pathol.
Pharfuacol., 93:101-107; Saito, S and I~ojima, Y, 1996 Comm.u~. Mol. Pathol.
Pharrnacol., 92: 119-126; Glennas, A. et aI, I986 Biochern. Pharnzacol.,
35:2033-40).
Cisplatinum, one of the most commonly prescribed chemotherapeutic agents,
contains
the heavy metal platinum. It has activity against diverse solid tumors
including
prostate, bladder, head and neck, ovarian, and lung cancers. However,
cisplatin
therapy is dosed to avoid acute dose-dependent nephrotoxicity in patients.
More
clinically significant is the limitation of the duration of cisplatin therapy
due to
cumulative nephrotoxicity. Both of these therapeutic heavy metals are limited
in
acute dose selection and duration of chronic therapy by nephrotoxicity based
in the
renal tubules (Ramesh, G. and Reeves, WB. 2002 J. Clifz. Invest., 100:835-42;
Boogaard, PJ et al, 1990 Co~trib. Nephrol., 83:208-12).
Genetic mutations of metallothionein, leading to reduced copper binding, are
also
implicated in several copper storage diseases (Suzulei-I~urasalei et al, cited
above;
Olcabe, M. et al, "Relationship between Cu ynetabolisyn hereditary disorders
a~zd
distribution of Czd nzetallothioszein irr kidneys" in Metallothionein IV,
I~laassen, CD
(ed), 1999 Birkhauser Verlag, Basei, GE pp413-419).
In all of these cases, the major site of damage caused by exposure to heavy
metals is the proximal tubules of the kidney. Free cadmium, copper and
cisplatinum
are toxic predominantly in the S3 proximal straight tubule (Sabolic, I. et al,
2002 Am.
J. Physiol. Renal., 283:F1389-F1402; Ramesh and Reeves, cited above; Okabe et
al,
cited above). In contrast, conjugation of the heavy metals to metallothionein
appears
to shift the damage to S~ and Sz sub-segments in the renal cortex (Young, IT.,
1977 J.
Histocheyfa., Cytochef~a. 25:935-941; Okabe et al, cited above).
2
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The different roles of intracellular and circulating MT have created much
confusion about the role of MT in cytotoxicity (Liu J. et al, 1994 Toxicol
Appl
Pharrnacol 128: 264-270; Nordberg M. 1984 Environ Health Perspect 54: 13-20;
Ramesh G and Reeves WB. 2000 JClin Invest 110: 835-842). Several lines of
evidence suggest that increased intracellular MT is a scavenger for heavy
metals,
providing protection against the effects of free heavy metals (Foullces EC,
''Role of
metallothionein in epithelial transport and seiluestration of cadmium." In:
Metallothiat~ein in Biology and Medicine, ed. I~laassen CD and Suzulci KT.,
Boca
Baton, FL: CRC, 1991, p. 171-182; Fowler BA et al, ''Proximal tubule cell
injury."
In: Nletallotlzionein in l3iolo~ y and O~ledicine, ed. Klaassen CD and Suzuki
KT. Boca
Baton, FL: CRC, 1991, p, 317-321; and Nordberg, cited above). This is one
basis for
the practice of administering bismuth to induce tissue MT clinically, before
administration of the heavy metal-based chemotherapeutic agent cisplatinum.
To design appropriate protective strategies against the heavy metal-caused
nephrotoxicity, the pathway by which metallothionein enters proximal tubular
cells
must be identified. Conjugation of heavy metals, such as copper or cadmium, to
metallothionein changes the nephron site of renal uptake of the heavy metals
and also
greatly enhances the nephrotoxic effect of these agents (Okabe M, et al,
''Relationship
between Cu metabolism hereditary disorders and distribution of Cu
metallothionein in
kidneys." In: M~tallothioneii~ Ih, ed, Klaassen CD. Basel: Birlcha~user
Verlag, 1999,
p. 413-419; Sabolic I, et a1, 2002. Arrr JPhysiol Renal Physie~l 283: F 1389-F
1402).
Changes in the nephron site of uptake of heavy metals follow conjugation to
metallothionein, when free Cu is taleen up in the inner medulla of mice.
However,
preconjugation of the Cu to metallothionein changed the uptake to the cortex
of the
leidney (Olcabe et al, cited above). A similar observation was made with
cadmium,
including morphological analysis of the nephron segments from S3 for un-
conjugated
cadmium to peri-glomerular S~ for metallothionein conjugated cadmium.
Metallothionein conjugation of cadmium was found to increase the tubular
toxicity
enormously (Sabolic et al, cited above).
The entry route of heavy metal-metallothionein complexes into the epithelial
cells remains unknown (Nordberg and Nordberg, 1987 J. LIOEH. 9 Suppl.:153-164;
CA 02554216 2006-07-19
WO 2005/072270 PCT/US2005/002023
Fowler, et al, "Proximal Tubule Gell Injury" in Metallothionein in Biology and
Medicine, Klassen, CD, Suzuki KT (eds)1, CRC Press, Boca Raton, FL, 1991:
pp311-
321; Liu et al, 2001 Toxicol. Appl. Plzarnaacol., 175:253-259; Endo et al,
2000
Toxicol., 146: 187-95; Kroning, et al, 1999 Br. J. Cancer; 79:293-299; Sharma
and
McQueen, 1982 Biochef7a. Pharmacol., 31:2153-9; Kone et al, 1990 J. Membr.
Biol.,
113:1-12). Conflicting reports implicate different transporters or receptor
mediated
pathways (Foulkes, cited above; Bernard et al, 1988 Kidney Irrt., 34:185-85;
Bernard
et al, 1988 Toxicol. Appl. Pharmacol., 87:440-5; Tsuruoka et al, 2000 J.
Plaarn~acol.
Exp. Ther., 292:769-77; Kinne et al, 1995 Toxicol., Appl. Pharn2acol, 1356:216-
21;
Marshall et al, 1994 J. Subrnicrosc. Cytol. Pathol., 26:59-66). At least some
of the_
uncertainty arises from the use of in vivo and irr vitro models that differ
significantly
in their behavior. For example, while CdCl2 is more toxic than Cd-
metallothionein to
cultured rat kidney proximal tubules and LLC-PK1 cells, Cd-metallothionein
shows
greater in vivo nephrotoxic effects (Liu et al, 1994 Toxicol. Appl.
Plzarmacol.,
128:264-70; Prozialeck et al, 1993 Life Sci., 53:PL337-42; and Sabolic et al,
cited
above). The lack of consensus complicates the search for a therapy for renal
heavy
metal poisoning regardless of the source of the metal.
There is a clear need in the art for an appropriate therapy that permits
urinary
excretion of cadmium, secondary to inhibition of renal proximal tubular
uptalee of Cd
or Cu metallothionein in the treatment of cadmium and copper storage diseases,
and
to eliminate the toxic accumulation of this heavy metal from environmental
sources.
There is also a need in the therapeutic uses of heavy metals for inhibition of
renal
uptake of the metals to enable the broadening of dose selection and treatment
duration
thereof.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a pharmaceutical composition
useful for the treatment of cancer which includes a conjugate formed by a
modified
metallothionein (MT) amino acid sequence or fragment thereof that binds the
megalin
receptor less avidly than naturally-occurring metallothionein and multiple
molecules
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WO 2005/072270 PCT/US2005/002023
of a therapeutic divalent metal ion. In one embodiment the modified MT does
not
bind megalin.
In another aspect, the invention comprises a composition formed by the
described conjugate which further includes a delivery peptide for targeted
delivery to
a desired cell, wherein said delivery peptide is fused to the modified MT or
fragment
thereof.
In another aspect, the invention includes a method for treating cancer
comprising administering to a mammalian subject an effective amount of the
pharmaceutical composition described herein. This method inhibits the renal
uptake
of divalent metal ions.
Another aspect of this invention is the use of the above-defined compositions
in the preparation of a medicament for the treatment of cancer.
Still another aspect of this invention includes a method for inhibiting renal
uptake of therapeutic divalent metals ions by administering the metal ions in
admixture with a conjugate of the above-defined composition.
In yet a further aspect, the invention provides a method of manufacturing an
above-defined conjugate by synthesizing a modified MT amino acid sequence or
fragment thereof that does not bind megalin with zinc divalent ions complexed
thereto; lowering the pH of the synthesized product to remove zinc therefrom;
dialyzing the synthesized product and contacting it with the selected
therapeutic
divalent metal ion; and either raising the pH to complex the selected metal
ion to the
sequence or incubating with the selected metal.
Yet a further aspect of this invention includes a metallothionein derivative
amino acid sequence or fragment thereof that does not bind megalin as avidly
as
naturally occurring metallothionein, the sequence optionally binding metal
ions.
These sequences are not taken up by cells of the kidney, including proximal
tubular
cells, cells of the ear or inner ear, or other cells of the body. In one
embodiment, such
metallothionein derivatives are mutated at the highly conserved hinge or
interdomain
region, centered on a lysine repeat, so that such derivatives bind heavy
metals but do
not bind megalin and thus are not taken up by proximal tubular cells.
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Other aspects and advantages of the present invention are described further in
the following detailed description of the preferred embodiments thereof.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the sequence of metallothionein class :I (MT) SEQ ID NO: 1,
indicating cadmium (Cd) binding sites and a and (3 domains below the sequence.
Aligned peptide fragments of SEQ ID NO: 1 used in interference studies assayed
by
surface plasmon resonance (SPR) techniques are indicated above the MT
sequence.
The lysine repeat that appears in the hinge region is indicated.
FIG. 2 indicates the results of surface plastnon resistance (SPR) analysis of
the
dose-dependent binding of rabbit kidney MT in HEPES-buffered saline (HBS)
containing 2 mM Ca and Mg to megalin. A graph plotting resonance units (RU)
vs.
MT molar concentration shows fit of the maximum responses obtained after 2.5
minutes. The double-referencing method of(Myszka DG. 1999 ,JA~IoI Recog~7it
12:
279-284) was used to eliminate artifacts in the data.
FIG. 3 is a bar graph indicating the flow cytometry analysis of the
displacement of fluorescently labeled MT from brush-border membranes by anti-
receptor antisera. Brush-border membrane vesicles isolated from rat renal
cortex were
incubated with fluorescently conjugated MT and receptor antisera. The observed
fluorescence is shown for the control (MT alone) and MT in the presence of
anti-
cubilin, anti-megalin, and anti-NI~1-peptide antibodies. Data files of 2,000
observations/sample were collected.
FIG. 4 is a bar graph of the flow cytometry analysis employed to show the
displacement of fluorescently labeled MT from brush-border membranes by
peptides
and ligands. Values are means ~ SE. Brush-border membrane vesicles isolated
from
rat renal cortex were incubated with fluorescently conjugated MT and various
peptides and ligands. The observed fluorescence is shown for the control (no
MT),
and fluorescent MT alone (MT-no competition), as well as the effect of added
peptide
2 (amino acids 10-26 of SEQ ID NO: 1), overlap peptide SCI~KSCC (amino acids
28-34 of SEQ ID NO: I), lenown megalin ligand (32-microglobulin, and
antibodies to
the ATI receptor (ATl R; a control for nonspecific binding), and competition
with
6
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equimolar concentration of unlabeled MT (M.T-competition). Data files of 2,000
observations/sample were collected; n = 6.
FIG. ~A is a graph ShOW111g the tlow cytometry analysis ofthe dose
dependence of tluorescently labeled MT into BN-16 cells. Concentration of MT
producing halt=maximal uptake is 4-8 ~M. In view ofthis data, all later
experiments
used at least 4 yM MT (10 pM preferred when reagents were not limiting).
FIG. SB is a graph showing the flow cytometry analysis of the time
dependence of fluorescently labeled MT into BN-16 cells. Nearly linear uptake
of
MT-FLUORX was observed over 3+ h at 2 doses. As a result, uptake experiments
with antibodies and MT-Cy3 were performed for l-2h.
FIG. SC is a graph showing competitive uptake of fluorescently labeled MT
into BN-16 cells. Known megalLn ligand (32-microglobulin displayed dose-
dependent
interference with MT uptake over a broad range of concentrations of bath (32-
microglobulin and MT.
FIG. 6A is a bar graph showing flow c5~tometry analysis of antibody inhibition
of uptake offluorescently labeled MT into BN-1G cells. Anti-cubilin antisera
inhibited
MT uptake into BN-16 cells in a concentration-dependent manner. The effect of
anti-
megalin (meg) antiserum was far greater than anti-cubilin (cub) antiserum; the
2 sera
produced an additive effect. Anti-A'1'1R antiserum, which also binds BN-16
cells, was
used as a n0115peC1fiC binding control but had no effect on MT uptake.
FIG. 6B is a bar graph showing flow cytometry analysis of peptide inhibition
of uptake of fluorescently labeled MT into BN-16 cells. Peptide ~, containing
the
overlap sequence SGKKSCC (amino acids 28-34 of SEQ I'D NO: 1) inhibited. MT
uptake, as did the overlap sequence itself. In contrast, pe~ticr'e 2, distant
from the
overlap sequence but with heavy cysteine content, did not affect MT uptake.
Concentrations of all peptides were 1.00 ~M.
FIG. 6C is a bar graph showing flow eytometry analysis of recombinant
(recomb) protein inhibition of uptake of fluorescently labeled MT into BN-16
cells.
Recombinant (recomb) full-length mouse MT Lnhibited the uptake of
fluorescently
labeled MT by BN-16 cells, as did the a-subunit carrying the intact SCKI~SC.C
7
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(amino acids 28-34 of SEQ ID NO: 1) motif The (i-subunit, in which this motif
is
disrupted, was far less effective at inhibiting MT uptake.
FIG. 7 is a bar graph indicating the effects on renal function in mice treated
with a control, with cisplatin alone (cisplat), with a complex formed of MT
and zinc
(ZnMT), with a complex formed of MT and cisplatin (MT-cis), a complex of this
invention formed of the MT-a subunit only with cisplatin (alpha-cis) and a
complex
of this invention formed of the MT-(3 subunit with cisplatin (beta-cis) as
discussed in
Example 8 below.
FIG. 8 is a bar graph illustrating the results of a 4 hour uptalee into DMS53
small cell lung cancer cells of phosphate buffered saline (PBS), cisplatin
alone (cis),
metallothionein alone (MT), a complex formed of MT and cisplatin (MT-cis), a
complex of this invention formed of the MT-a subunit only with cisplatin
(alpha-cis)
and a complex of this invention formed of the MT-/3 subunit with cisplatin
(beta-cis)
as discussed in Example 8 below.
FIG. 9 is a bar graph indicating the results of a Caspase-3 induction assay of
MT and complexes of MT subunits and cisplatin in into DMS53 small cell lung
cancer cells as discussed in Example 8 below. The symbols are as stated in
FIG. 8.
FIG. 10 is a bar graph illustrating the results of a 4 hour uptake into J82
transitional cell bladder cancer cells of phosphate buffered saline (PBS),
cisplatin
alone (cis), metallothionein alone (MT), a complex formed of MT and cisplatin
(MT-
cis), a complex of this invention formed of the MT-a subunit only with
cisplatin
(alpha-cis) and a complex of this invention formed of the MT-~3 subunit with
cisplatin
(beta-cis) as discussed in Example 8 below.
FIG. 11 is a bar graph indicating the results of a Caspase-3 induction assay
of
MT and complexes of MT subunits and cisplatin in J82 transitional cell bladder
cancer cells as discussed in Example 8 below. The symbols are as stated in
FIG. 10.
FIG. 12 is a bar graph indicating ovarian cancer OVCAR3 cell uptake of
metallothionein. OVCAR3 cells grown to confluence in 96 well plates were
exposed
to 40pM Cy3-fluorescently conjugated MT for 1,2 and 4 hours. Cells were
washed,
surface binding released with acid, and trypsinized for flow cytometry
analysis of MT
uptake.
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FIG. 13 is a bar graph showing that cisplatin and MT-complexed cisplatin kill
OVCAR3 cells. OVCAR3 cells grown to confluence in 96 well plates were exposed
to varied concentrations of cisplatin or MT-complexed with cisplatin and cell
death
assayed by trypsinizing the cells off the beads, filtering off the beads, and
staining
with propidium iodide for membrane integrity. Propidium iodide uptake was
assayed
by flow cytometry.
FIG. 14 is a graph showing time course of caspase-3 activity after cisplatin
treatment of J82 transitional carcinoma cells, using cisplatin alone or
cisplatin
conjugated to the (3-subunit of MT. Values shown are mean ~ standard error,
n=6.
Data files of 2,000 observations/sample were collected by flow cytometry.
DETAILED DESCRIPTION OF THE INVENTION
In response to the need of the art for additional therapeutic compositions and
methods for the treatment of cancer with heavy metal therapeutics and for the
treatment of heavy metal poisoning, the inventors identified the receptor in
the
proximal kidney tubules to which naturally occurring MT (optionally laden with
heavy divalent metal ions) binds. The binding between the MT complexed with a
heavy metal ion and megalin causes the MT-heavy metal complex to remain in
high
concentration in the renal tubules, resulting in nephrotoxicity and renal
tubule
damage. Using this discovery, the inventors then provided the following
compositions for meeting the above-identified needs of the art.
A. Tlae Metallothioueiu Receptor iu the Reual Tubules
The inventors have determined that megalin binds metallothionein and is the
most important mechanism of MT uptake in to the renal proximal tubule. The
inventors have further identified the highly conserved hinge or interdomain
region of
metallothionein, centered on a lysine repeat, as the critical site for binding
to megalin.
(1) Ideyztificc~tiotz of the Receptor
Megalin is the most abundant protein in the proximal tubule of the
kidney. It is heavily expressed in Si in the peri-glomerular area, and is a
mufti-ligand
receptor with multiple binding sites. Megalin has 4 binding sites, and cubilin
at least
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27 domains and 8 EGF repeats as binding sites, and yet these two proteins are
thought
to be largely responsible for the reabsorption of an immense volume of diverse
ligands in the proximal tubule (Saito A et al, 1994 Proc Natl Acad Sci USA 91:
9725-
9729; Verroust PJ. et al, 2002 Kidney Int 62: 745-756, Verroust PJ and
Kozyraki R.
2001 Curr Opirr Neplarol Hypertens 10: 33-38). Given the long list of ligands
for
megalin, and the abundance of these proteins in the glomerular filtrate, the
effectiveness of the uptake likely relies on the very large content of megalin
in the
kidney (Verroust PJ and Kozyraki R. 2001 Cm°r Opin Neplzrol Hyperter~s
10: 33-38).
On simple SDS-PAGE gels of renal proximal tubular brush borders, the two most
abundant proteins are the distinctive 460and 600-kDa molecular masses of
cubilin and
megalin (Verroust PJ. et al, 2002 Kidney Int 62: 745-756). When one combines
the
abundant expression of megalin with the large surface area created by brush-
border
formation, there is abundant megalin to facilitate reabsorption of all
available ligands
(Verroust PJ. et al, 2002 Kidney Int 62: 745-756, Verroust PJ and Kozyraki R.
2001
Curr Opin Nepl7rol Hyperteyis 10: 33-38).
Experiments described in the examples below support the
determination that binding of MT to megalin is critical in renal proximal
tubular
uptake of MT-bound heavy metals. First, MT binds megalin, but not cubilin, in
direct
surface plasmon resonance (SPR) studies in a dose, ion and pH dependent
manner.
Binding of MT occurs at a single site with a Kd 10-4 and, as with other
megalin
ligands, depends on divalent canons. Second, antisera and various known
megalin
ligands inhibit the uptake of fluorescently labeled MT in model cell systems.
Anti-
megalin antisera, but not control sera or anti-NK-1 receptor, displace >90%
bound
MT from rat renal brush-border membranes. Megalin ligands, including (32-
microglobulin and also recombinant MT fragments, identified below, compete for
uptake by megalin-expressing rat yolk sac BN-16 cells. Third, megalin and
fluorescently labeled MT colocalize in BN-16 cells, as shown by fluorescent
microscopic techniques.
The distribution and molecular characteristics of megalin are consistent with
these results of metallothionein uptake. Taken together with the observation
that
megalin mediates uptake of ~32anicroglobulin, these result provides evidence
CA 02554216 2006-07-19
WO 2005/072270 PCT/US2005/002023
implicating megalin in metallothionein uptake. The results illustrated in the
examples
and data herein are also consistent with the bmdmg properties and
physiological
observations of megalin and its ligands in other systems. The dissociation
constant
estimated at 9.8 x 10 -S M may appear small for a receptor-ligand interaction,
but it is
similar to values obtained for other known megalin ligands (Gburek J, et al,
2002 J
Ar~r Soc Neplar°e~l 13: 423-430). The calcium dependence of MT binding
is also
consistent with similar ion requirements of other megalin ligands (Birn H, et
al, 1997
JBiol Cher~~ 272: 26497-26504; Moestrup SK. et al, 1998 .I Biol Ch~rrr 273:
5235-
5242), but the dependence on magnesium is unusual. The inventors' observation
of
competition between /32-microglobulin and MT binding to megalin establishes
that
MT and ~2-microglobulin compete directly for renal uptake in live animals
through
competition for megalin binding. Diverse MTs bind megalin with the same
kinetics
and may be important to ensure efficient reuptale of diverse isoforms in the
proximal
tubule.
(2) Iden.tificatiorr of the Receptor'-MT C'~itical Binding Site
The initial analysis of the MT/megalin binding site used a peptide
library, because the midregion of the MT molecule models to a linear peptide
with
little secondary or tertiary structure. MT is so highly conserved across
species and
phyla that comparison of protein isoforms from different species (even the
naturally
occurring isoforms of MT from Ia and Ib, through II, III, IV, V, and others)
was not
useful in defining binding sites (Atrian S, et al., "Recombinant synthesis and
metal-
binding abilities of mouse metallothionein 1 and its a-and ~3-domains." In:
Metallothiorrein II ; ed. Klaassen CD. Basel: Birkha"user Verlag, 1999, p. 55-
61;
Huang PC et al, "Native and engineered metallothioneins." In:
Metallotlrio>zeirr ire
Biology and Medicine, ed. Klaassen CD and Suzuki KT., Boca Raton, FL: CRC,
1991, p. 87-101; and Kagi JHR and Vasalc M., "Chemistry of mammalian
metallothionein." In: Metallotlriohein ire Biology arid Medicine, eds.
IClaassen CD and
Suzuki KT., Boca Raton, FL: CRC, 1991, p. 49-60.)
Using follow-up SPR and flow cytometry studies with overlapping MT
peptides and recombinant MT fragments, the inventors identified the hinge
region
SCKKSCC (aa 28-34 of SEQ ID NO: 1) of MT as one critical site for megalin
11
CA 02554216 2006-07-19
WO 2005/072270 PCT/US2005/002023
binding. In lower species, MT exists as two separate molecules, binding three
and
four heavy metal moieties. However, in mammals and other higher organisms the
two
molecules have coalesced, joined by a hinge region centered on a highly
positively
charged lysine repeat. The hinge region sequence SCKKSCC (aa 28-34 of SEQ ID
NO: 1 ) is even more heavily conserved than the rest of the MT sequence, being
identical in virtually all known mammalian species, and all the various MT
isoforms
in each species (Huang et al, cited above; Kagi and Vasak, cited above).
Studies
using site-directed mutagenesis have established the critical role of the
conserved
lysine repeat of the hinge region in the detoxification function of MT in
yeast.
Replacement ofone or both Iysines in the hinge or interdomain region was
inconsequential to the structure and function of MT unless both substituted
residues
are uncharged (Cody GW and Humg PC. 1993 Bioclm~r~istf~a 32: 5127-5131; Cody
CW and Huang PC. 1994 Biocher3~ Biophys Res C'0779Y72ZlYl 202: 954-959).
Numerous
investigators reported extremely low yields and splice variants when
attempting to
express MT (Atrian, cited above), thereby implying that site-directed
mutagenesis and
expression of mutated MT isoforms was not likely to be useful.
After the failure of non-MT-derived peptides with a central KK motif to
inhibit megalin-MT interaction in direct SPR studies ofthe examples implicated
the
interdomain of MT as a binding site, the inventors investigated recombinant MT
subunits (Atrian et al), which disrupt the candidate binding motif.
Recombinant
production of MT fragments dividing MT at the lysine-lysine hinge yields
intact a-
and (i-subunits that still bind heavy metals (Atrian, cited above). The
inventors'
observations of charged peptides and the highly charged polybasic antibiotic
gentamicin suggest more structural requirements than simply charge for a
molecule to
interfere with the megalin-MT interaction.
The following compositions and methods of this invention are based on the
discovery that disruption of the SCKKSCC motif inhibits proximal tubular MT
uptake
and thereby eliminates much of the renal accumulation and toxicity of heavy
metals,
such as cadmium, gold, copper, and cisplatinum.
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B. Compositions of tlae Iizve~ztio~a
Iii view of the above-noted discovery of the MT receptor and binding site i~
vivo, the inventors have determined that mutants of metallothionein that bind
heavy
metals, but do not bind megalin, or that bind megalin with reduced avidity
compared
to MT, are not taken up by proximal tubular cells. Thus, such mutant MTare
useful in
the field of heavy metal therapeutics. More specifically, the inventors
provide herein
compositions that can deliver a therapeutic heavy metal, e.g., preferably anti-
neoplastic platinum compounds, on a mutated MT which does not bind megalin.
This composition and uses thereof avoid therapy-limiting nephrotoxicity of
heavy
metals used as anticancer agents.
A composition of the present invention is a conjugate formed by (1) a
modified metallothionein (MT) amino acid sequence or fragment thereof that
binds
the megalin receptor less avidly than naturally-occurring MT and that binds a
divalent
metal ion or heavy metal molecule and (2) multiple molecules of a therapeutic
divalent metal ion.
With regard to the modified MT sequence of this conjugate, the term "binding
less avidly than naturally-occurring MT" as used herein is defined as a
binding
affinity between the modified MT and megalin of less than Kd ~10-5 to a Kd of
0.
Such a binding affinity is low enough that the naturally-occurring MT ih vivo
would
successfully compete with the modified MT for binding to megalin. Such a low
binding affinity also includes no binding of the modified MT amino acid
sequence of
this invention to megalin receptor at all. More specifically, any modification
to MT
sequences that involves disruption of the SCKKSCC hinge region sequence of
naturally-occurring MT will provide a sequence that meets the requirements of
this
invention.
(1) Modified Full-Length MT Sequences of the Invet~tiofz
Thus, an exemplary modified MT sequence is either a full-length MT
sequence or modified full-length sequence of naturally-occurring MT in which
the
hinge region is disrupted. For example, one embodiment of a modified MT of
this
invention includes a sequence of formula
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MDPNC~SC2ATGNSC3TC4ASSCSKC6KEC~KC8TSC9X X'SC~aCi~SCI2C~3PAGC,4
TKC~SAQGC~6IC»KGASDKC~$SCl9CaoA, SEQ ID NO: 4, wherein X and X' are
independently selected from any amino acid other than K. In another embodiment
includes the sequence of SEQ ID NO: 4, in which X and X' are any uncharged
amino
acids. In another embodiment includes the sequence of SEQ ID NO: 4, in which X
and X' are any negatively charged amino acids. Still another embodiment of a
modified MT of this invention includes SEQ ID NO: 4 in which in which X and X'
are non-naturally occurring amino acids, as defined below. Still another
embodiment
of a modified MT of this invention includes SEQ >I7 NO: 1 in which in which X-
X'
include a spacer or bridge of one or more naturally-occurring or non-naturally-
occurring amino acids, carbohydrate moieties or other chemical moieties
interposed
between the amino acid residues of X and X', wherein X and X' may be any amino
acid, including lysine. The spacer or bridge is of any size or composition
sufficient to
disrupt the positive charge formed by the KK sequence.
Preferably, any modification of the KK sequences that disrupts the strong
positive charge at that position in the MT sequence will provide a
modification
sufficient to prevent binding of the modified MT to the megalin receptor. For
example, modifications that place a negative charge on the MT sequence at that
point
are useful. Modifications of the full-length MT sequence that convert the
positive KK
cloud at the hinge region into a neutral 'charge' are also useful.
Additionally, any modification of the MT sequence that enlarges the complex
formed by the MT bound to the heavy metal molecules to a size that cannot fit
through the renal glomeruli is useful for this purpose. Preferably the size of
the
modified MT in this embodiment is larger than the ~701cD sieves of the kidney.
Thus,
included in the modification of full-length MT includes conjugates of the
modified
full-length MT, such as multiple modified full-length MT fused together to
form a
larger protein sequence. Also included as modifications full-length MT are
large
proteins formed by modified full-length MT which are modified by chemical or
carbohydrate moieties to form a large protein that cannot fit through the
renal
glomeruli. Molecules that do not fit through the renal glomeruli will be
filtered
through the liver rather than the leidney. Such compounds are not toxic to the
liver.
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WO 2005/072270 PCT/US2005/002023
A further modified MT sequence includes SEQ ID NO: 4 in which X and X'
are selected from any of the embodiments above and in which all cysteine
residues are
invariant, and further in which any of the non-C residues are substituted by
non-
naturally occurring amino acids, as defined below. Such non-naturally
occurring
amino acids can contribute to changing the charge or size of the modified full-
length
MT, to fit the parameters described above.
In other embodiments of a modified full-length MT sequence of this invention,
and in any of the embodiments described above, X and X' are both glutamine
residues.
(2) Modified F~~agnae~ts of MT Sequences of the I~vehtiofz
Another exemplary modified MT sequence is a fragment of the naturally-
occurring MT sequence in which the hinge region is disrupted. As one example,
a
modified MT sequence comprises a modified (I-MT subunit sequence of the
formula
MDPNCISCZATGNSC3TC4ASSCSKC6KEC~KC8TSC9X SEQ ID NO: 2, wherein X
is any amino acid other than K. In another embodiment, the MT sequence is SEQ
ID
NO: 2, wherein X is an uncharged amino acid other than K. In still another
embodiment, X can be a negatively-charged amino acid. In still another
embodiment, the MT sequence is SEQ ID NO: 2, wherein X is a non-naturally
occurring amino acid, as defined below. In yet a further embodiment, the
modified
MT sequence is SEQ ID NO: 2 in which X is selected from any of the embodiments
above and in which all cysteine residues are invariant, and further in which
any of the
non-C residues are substituted by non-naturally occurring amino acids, as
defined
below.
Yet a further embodiment of a modified MT fragment according to this
invention includes SEQ ID NO: 2, in which X is defined as any of the above-
noted
suggestions and which is truncated at the amino terminus, leaving as the first
amino
terminal amino acid, the residue C~, A further embodiment of a modified MT
fragment according to this invention includes SEQ ID NO: 2, in which X is
defined as
any of the above-noted suggestions and which is truncated at the amino
terminus,
leaving as the first amino terminal amino acid, the residue CZ, A further
embodiment
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WO 2005/072270 PCT/US2005/002023
of a modified MT fragment according to this invention includes SEQ ID NO: 2,
in
which X is defined as any of the above-noted suggestions and which is
truncated at
the amino terminus, leaving as the first amino terminal amino acid, the
residue C3, A
further embodiment of a modified MT fragment according to this invention
includes
SEQ ID NO: 2, in which X is defined as any of the above-noted suggestions and
which is truncated at the amino terminus, leaving as the first amino terminal
amino
acid, the residue C4,
Other fragments of this invention may include the ~i subunit of MT, truncated
at the carboxy terminus. Truncations of the subunit must be sufficient to
permit at
least one divalent heavy metal to be carried by the modified MT fragment.
Another exemplary modified fragment of the naturally-occurring MT
sequence in which the hinge region is disrupted includes a modified a - MT
subunit
sequence of the formula
X'SG1oC11SC12C13PAGC1~TKC15AQGC~6IC1~KGASDKC1$SC19C2oA, SEQ ID NO:
3, wherein X' is any amino acid other than K. In another embodiment, the MT
sequence is SEQ ID NO: 3, wherein X' is an uncharged amino acid other than K.
In
another embodiment, the MT sequence is SEQ ID NO: 3, wherein X' is a
negatively
charged amino acid. In still another embodiment, the MT sequence is SEQ ID NO:
3, wherein X' is a non-naturally occurring amino acid, as defined below. In
yet a
further embodiment, the modified MT sequence is SEQ ID NO: 3 in which X' is
selected from any of the embodiments above and in which all cysteine residues
are
invariant, and further in which any of the non-C residues are substituted by
non-
naturally occurring amino acids, as defined below.
Yet a further embodiment of a modified MT fragment according to this
invention includes SEQ ID NO: 3, in which X' is defined as any of the above-
noted
suggestions and which is truncated at the carboxy terminus, leaving as the
last
carboxy terminal amino acid, the residue C2o, A further embodiment of a
modified
MT fragment according to this invention includes SEQ ID NO: 3, in which X' is
defined as any of the above-noted suggestions and which is truncated at the
carboxy
terminus, leaving as the last carboxy terminal amino acid, the residue C~9, A
further
embodiment of a modified MT fragment according to this invention includes SEQ
ID
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WO 2005/072270 PCT/US2005/002023
NO: 3, in which X' is defined as any of the above-noted suggestions and which
is
truncated at the carboxy terminus, leaving as the last carboxy terminal amino
acid, the
residue C~B. A further embodiment of a modified MT fragment according to this
invention includes SEQ ID NO: 3, in which X' is defined as any of the above-
noted
suggestions and which is truncated at the carboxy terminus, leaving as the
last
carboxy terminal amino acid, the residue C1~. A further embodiment of a
modified
MT fragment according to this invention includes SEQ ID NO: 3, in which X' is
defined as any of the above-noted suggestions and which is truncated at the
carboxy
terminus, leaving as the last carboxy terminal amino acid, the residue C~6,
Modified
a-MTs of this invention may also include the sequences truncated at the amino
terminus. Truncations of the subunit must be sufficient to permit at least one
divalent
heavy metal to be carried by the modified MT fragment.
Still other embodiments of a modified aMT or ~3MT fragment according to
this invention includes any of the above embodiments, in which any of the non-
C
residues are substituted by non-naturally occurring amino acids, as defined
below.
In other embodiments of a modified a or (i MT fragment of this invention, and
in any of the embodiments described above, X or X' is a glutamine residue.
Still other embodiment of a modified MT fragment according to this invention
includes any of the above embodiments of the modified a- or J3-MT subunits, in
which
any of the non-C residues are substituted by non-naturally occurring amino
acids, as
defined below. Still other fragments of these modified subunits are those in
which
one or more amino acids are attached to chemical or carbohydrate moieties
sufficient
to increase the size of the fragment to larger than will be filterable by the
leidneys, or
that will retain a neutral or negative charge. As described above, among such
additional modifications are conjugates of multiple copies of the (3-subunit
of MT,
multiple copies of the a-subunit of MT, or conjugates of one or more copies of
the (3-
subunit of MT with one or more copies of the full-length modified MT, or
conjugates
of one or more copies of the modified [3-MT with one or more copies of the a-
MT, or
conjugates formed by one or more copies of the modified (3-MT with one or more
copies of the modified a-MT and one or more copies of the modified full-length
MT.
Such conjugates may be formed of each modified MT sequence or fragment fused
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WO 2005/072270 PCT/US2005/002023
directly to a terminal amino acid of another modified MT sequence or fragment.
Such
conjugates may be formed of each modified MT sequence or fragment fused
indirectly to another modified MT sequence or fragment via a chemical, amino
acid or
carbohydrate linker. Desirably, such conjugates are greater in size than the
size of the
kidney sieves, e.g., greater than 70kD in size when coupled with the
appropriate
heavy metals.
(3~ Natuf°ally and Non-Naturally Occurri~rg Ana.irzo Acids
"Naturally-occurring amino acid" is used herein to refer to the twenty amino
acids that occur in nature in L form, which include alanine, cysteine,
aspartate,
glutamate, phenylalanine, glycine, histidine, isoleucine, lysine, leucine,
methionine,
aspargine, proline, glutamine, arginine, serine, threonine, valine,
tryptophan, and
tyrosine, or any derivative thereof produced through a naturally-occurring
biological
process or pathway.
"Non-naturally-occurring amino acid" is used herein to refer to an amino acid
other than a naturally-occurring amino acid as defined above, which can be
synthesized or "man-made", and including a derivative thereof, whether
produced
synthetically or via a biological process or pathway. Non-naturally occurring
amino
acids include, without limitation, D amino acids, amino acids containing
unnaturally
substituted side chains, e.g., methyl-Arg, cyclic amino acids, diamino acids,
l3-amino
acids, homo amino acids. Non-naturally-occurring or unnatural amino acids may
be
characterized by novel backbone and side chain structures and are widely
available
from commercial reagent suppliers, such as Sigma-Aldrich
(www.si~naaldrich.com),
www.Netchem.com and other sites. See also a broad literature on such
structures
including, without limitation, Han S and Viola RE, Protein Pept. Lett. 2004
11(2):104-14; Ishida et al, Biopolymers 2004 76(1):69-82; Sasaki et al, Biol.
Pharm.
Bull. 2004 27(2):244-7; Pascal R et al, Meth. Enzymol. 2003 369:182-94; Yoder
NC
and Kumar IC, Chem. Soc. Rev. 2002 31(6):335-41; and Ager DJ, Curr. Opin. Drug
Discov. Devel. 2002 5(6):892-905, among others, which are incorporated herein
by
reference. This term does not encompass those derivatives which fall within
the
definition of a "naturally-occurring amino acid", as defined above.
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WO 2005/072270 PCT/US2005/002023
Such non-naturally occurring amino acids) when employed in the compounds
above are anticipated to make the compounds more resistant to degradation by
mammalian enzymes in serum, saliva, stomach and intestines, and thus compounds
that are composed of one or more such amino acids may confer upon the compound
enhanced stability and bioavailability in vivo. A variety of methods for
producing
non-natural amino acids are known and may be selected by one of skill in the
art.
For example, one class of non-naturally occurring amino acids are L amino
acids that
effect stereochemistry. Thus, in one embodiment of compounds of this
invention, one
or more of the amino acids in the peptide may be in L form, while others may
be in D
form. Another non-naturally occurring amino acid is an amino acid which is
modified
to contain a substitution on the alpha-carbon in the amino acid structure. For
example
the alpha-carbon may be substituted by a suitable hydrocarbon moiety, such as
aminoisobutyrate. Still another class of non-naturally occurring amino acids
is amino
acids which are modified or mutated to extend their carbon chain length. For
example, an amino acid with a single alpha-carbon chain, may be extended with
at
least one additional carbon, i.e., a beta-carbon, and so on. An additional
modification
to an amino acid is the insertion of a substituent on the nitrogen of the
amino group.
An example of this type of modification is an N-methyl amino acid. The
addition of
substituents on the alpha carbon or additional carbons or on the nitrogen of
the amino
acid molecule may occur in any of the amino acids of the formula above.
Among useful substituents for creating the non-naturally occurring amino
acids are a straight chain, branched, cyclic or heterocyclic GI_12 alkyl
group, and
straight chain, branched, cyclic, or heterocyclic C1_12 alkanoyl group. The
amino acid
may be also modified by the insertion of modifying sugars, imide groups and
the like.
Other amino acids are substituted in the ortho or meta position by a
substituent such
as H, OH, CH3, halogen, OCH3, NH2, CH or NO2.
A non-exclusive list of modified or non-naturally occurring amino acids for
inclusion in compounds fitting the formula above include amino acids modified
by N-
terminal acetylation, C-terminal amidation, fonnylation of the N-terminal
methionine,
gamma-carboxyglutamic acid hydroxylation of Asp, Asn, Pro or Lys residues in
the
compound, methylation of Lys or Arg, preferably; phosphorylation of Ser, Thr,
Tyr,
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WO 2005/072270 PCT/US2005/002023
Asp or His in the compound, use of a pyrrolidone carboxylic acid, which is an
N-
terminal glutamate which has formed an internal cyclic lactam, sulfatation of
Tyr,
generally. Still other modifications of non-naturally occurring amino acids
include
use of or substitution with the following moieties: a 2-aminoadipic acid
group, a 3-
aminoadipic acid group, beta-Ala or beta-aminopropionic acid group, 2-
aminobutryic
acid, 4-aminobutyric acid, piperidinic acid, 6-aminocaproic acid, 2-
aminoheptanoic
acid, 2-aminoisobutryic acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,
4
diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic
acid, N-ethylglycine, N-ethylglycine, N-ethyl asparagine, hydroxylysine, allo-
hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-
isoleucine, N-
methylglycine, sarcosine, N-methylisoleucine, 6-N-methyllysine, N-
methylvaline, 6-
N-methyllysine, norvaline, norleucine, and ornithine.
One of skill in the art may readily select among such non-naturally-occurring
amino acids to modify the MT sequences or fragments to provide the modified MT
sequence or fragment with the appropriate or no binding affinity to megalin
according
to this invention.
A variety of publications directed to MT derivatives used in binding studies
propose modified MT sequences and fragments that may also be useful in the
methods and compositions of this invention. Such publications include, among
others, Tio et al, 2004, J. Biol. Chem., 279(23):24403-24413; Pan et al, 1999
Eur. J.
Biochem., 266:33-39; Romero-Isart et al, 1999 Eur. J. Biochem., 259:519-527;
Nielson and Winge, 1984 J. Biol. Chem., 259 (8):4941-4946; Nielson and Winge,
1985 J. Biol. Chem., 260(15):8698-8701; Valls et al, 2001 J. Biol. Chem.,
276(35):32835-32843, all incorporated herein by reference.
(5) Divalent Metal IOfTS o~ Heavy Metals
As a further component of compositions of this invention, the modified MT
sequence defined above are conjugated or associated with at least one or
multiple
molecules of heavy metals or divalent metal ions. Thus, compositions of this
invention desirably contain in association with the modified MT sequence from
one to
seven molecules of a divalent metal ion. In one embodiment, an MT fragment is
conjugated to three divalent metal ions. In another embodiment the MT fragment
is
CA 02554216 2006-07-19
WO 2005/072270 PCT/US2005/002023
conjugated to four divalent metal ions. Preferably a (3-MT fragment is
associated with
three metal ions. Preferably a a-MT fragment is associated with four metal
ions.
Where the modified MT sequences are conjugated together, the number of
molecules
of divalent metal ion are multiples of the one to seven molecules carried by
each
individual modified MT sequence.
Desirable divalent metal ions for such use include, without limitation, anti-
neoplastic platinum compounds, gold, copper, and cadmium. Known anti-
neoplastic
platinum compounds include cisplatin, oxyplatin and carboplatin. See, e.g.,
other
compounds disclosed in Platinum-Based Drugs in Cancer Therapy, eds. Kelland
and
Farrell (March 2000) ISBN Na. 1-59259-012-8, incorporated herein by reference.
Still other metal compounds that bind to the cysteine binding sites on
metallothionein
and are useful in therapeutic treatments of disease, e.g., cancers, are also
anticipated
to be useful in the compositions and methods of this invention. In one
specific
embodiment exemplified in Examples 7-8 below, a composition of this invention
includes a (3-MT fragment in which X is Q and which is associated with three
molecules of cisplatin. One of skill in the art, given this disclosure, may
readily
prepare other embodiments of this invention.
6. Optional Delivery Peptides
In other embodiments of this invention, the modified MT sequences
conjugated with divalent metal ions may be further associated With other
peptides or
proteins for the purpose of focusing the delivery of the conjugate to a
desired target
site. Therefore, in one embodiment, the composition includes a delivery
peptide for
targeted delivery to a desired cell, preferably a tumor cell. The delivery
peptide is
preferably fused to the modified MT or fragment thereof at one or the amino or
carboxy termini. Preferably the delivery peptide is attached at a point in the
MT
sequence that least disrupts the binding of the heavy metal molecules.
For example, such a delivery peptide may be a penetration enhancer or
transport sequence, a "cell penetrating peptide" (CPP) or "protein
transduction
domain" (PTD). Some examples of suitable CPPs are arginine-rich peptides, and
more specifically, linear or branched-chain peptides containing approximately
8
residues of arginine (See, e.g., Futalei et al Curr. Prot. Pept. Sci., 2003
4(2):87-96;
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WO 2005/072270 PCT/US2005/002023
and Futaki Int. J. Pharm, 2002 245(1-2):1-7, both incorporated by reference
herein).
Other suitable CPPs are also discussed in liiternational Published Patent
Application
Nos. WO 03/035892 and WO 03/035697.
Other delivery peptides for fusion to the compositions of this invention
include transactivating protein analogs or fragments thereof, such as the HIV-
1 Tat
(Vives et al, Curr. Protein Pept. Sci., 2003 4(2):125-32). The HIV-1 Tat basic
peptide
sequence is an example of the prototypic cell membrane-permeant component. US
Patent No. 6,348,185 refers to cell membrane-permeant peptides including
peptides of
4 to 6 amino acids derived from HIV-1 Tat, linked to pharmaceutically active
substances via a functional linker that confers target cell specificity to the
composition. U.S. Patent Nos. 5,804,604; 5,747,641; 5,674,980; 5,670,617;
5,652,122 (Frankel) refers to the use of Tat peptides to transport covalently
linked
biologically active cargo molecules into the cytoplasm and nuclei of cells.
Morris et
al, Nat. Biotec7znol., 2001 19(12):1173-76 refers to PTDs including TAT
protein
sequences. US Patent No. 5,804,604 refers to Tat-derived transport
polypeptides. A
commercial useful peptide transport molecule is the CHARIOTTM reagent (Active
Motif).
Still other options for the transport peptides useful in the present invention
are
described in U.S. Patent Nos. 5,135,736 and 5,169,933 (Anderson), which refer
to the
use of covalently linked complexes (CLCs) to introduce molecules into cells.
Yet
another embodiment of a delivery peptide is a peptide-oligodeoxynucleotide
conjugate described by L. Chaloin et al, Biochem., 1997 37:11179-87. These
conjugates comprise the combination of a peptide containing a hydrophobic
motif
associated with a hydrophilic nuclear localization sequence covalently linked
to a
small molecule to facilitate the cellular internalization of small molecules.
The
hydrophobic sequences used correspond to a signal peptide sequence or a
fragment of
the fusion peptide GP41. One peptide successfully targeted fluorescent
oligodeoxynucleotides into living cells (Chaloin et al, Biochem. Biophys. Res.
Commun., 1998 243(2):601-608). Still another suitable transport peptide is
described
by Taylor et al, Elect~opl~o~esis, 2003 24(9):1331-1337 and refers to an
amphipathic
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WO 2005/072270 PCT/US2005/002023
peptide Pep-1 which may be used as a transport peptide in combination with a
nonionic detergent carrier, for delivery of SDS-PAGE isolated proteins into a
cell.
The delivery or transport peptide useful in the present invention can be any
cell membrane-permeant basic peptide component of the complexes described in
the
above-cited documents, all of which are incorporated by reference herein. The
transport peptide can be a peptide or protein that comprises any amino acid
sequence
(including naturally-occurring amino acids or non-natural amino acids, such as
D
amino acids) that confers the desired intracellular translocation and
targeting
properties to the selected therapeutic peptide or protein. 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. Attachment of a compound of the formula of the present invention
to the
delivery peptide would permit the resulting composition to be localized within
cytoplasmic and/or nuclear compartments.
Specific delivery peptide sequences useful in practicing the present invention
include, but are not limited to, sequences of the following proteins and
fragments and
homologous sequences derived therefrom: the HIV-1 Tat protein, the HIV-1 Rev
protein basic motif, the HTLV-1 Rex protein basic motif, the third helix of
the
homeodomain of Antennapedia, a peptide derivable from the heavy chain variable
region of an anti-DNA monoclonal antibody, the Herpes simplex virus VP22
protein,
the ChariotTM protein, and the Pep-1 protein. The minimum number of amino acid
residues can be in the range of from about three to about six, preferably from
about
three to about five, and most preferably about four.
The delivery peptide sequence attached to the modified MT-metal complex
can also contain an enzymatic cleavage site fox interposition between the
delivery
peptide sequence and the MT sequence of the complexes described above. This
optional sequence permits the delivery peptide to be cleaved from the MT
sequence
intracellularly, if desired. Selection of such cleavage sites is within the
skill of the art.
Any of the compositions specifically identified herein and others within the
teachings of this specification can all be readily tested for the required
biological
function, e.g., the ability to bind megalin less avidly than naturally
occurring MT or
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WO 2005/072270 PCT/US2005/002023
not at all and the ability to complex with multiple molecules of divalent
metal ions, as
well as for the ability to deliver the metal ion to a target in mammalian
cells and
tissues ifa. vitro and in vivo. The resulting composition may be screened for
biological
activity and/or metabolic stability by in vitro and in vivo assays, such as
those
described in the examples and in the art. These compounds generally have
"significant" metabolic stability in mammalian serum, i.e., the compounds are
stable
for at least 2 hours in serum. More preferred compounds are stable for at
least 4 hours
in serum. Still more preferred compounds of this invention are stable in serum
for
greater than 8 hours.
C. Methods ofMauufacture
Compounds and conjugates of the invention may be prepared conventionally
by known chemical synthesis techniques. Compounds of the invention may also be
purchased from a commercial vendor, e.g. the Sigma-Aldrich Co. Among such
preferred techniques known to one of skill in the art are included the
synthetic
methods described by Merrifield, J. Amer. Chem. Soc., 1963 85:2149-2154; and
in
texts such as G. C. Barrett and D. T. Elmore, "Amino Acids and Peptides" Oct.
1998;
and "Peptides: Chemistry and Biology", eds. N. Sewald, H-D Jalcublce, Aug.
2002;
and other conventional textboolcs relating to the construction of synthetic
compounds.
Such compositions may be produced recombinantly by conventional methods.
Specific embodiments of compounds of this invention are disclosed in detail in
Example 7 below.
Such methods of producing and assembling the individual components of this
invention, as well as the conjugates and pharmaceutical compositions described
herein may use the techniques described in the examples or other techniques in
the
art. For instance, as demonstrated by Example 7, a method of producing a
composition as described above, a modified MT amino acid sequence or fragment
thereof that does not bind megalin is prepared by recombinant methods or
purchased.
Thereafter, the MT sequence was combined in a suitable ratio in a suitable
buffer and
incubated for a time sufficient to replace any divalent metal ions on the MT
with the
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CA 02554216 2006-07-19
WO 2005/072270 PCT/US2005/002023
desired metal ion. In an alternate step, the selected metal ion may be
complexed with
the modified MT by raising the pH to complex the selected metal ion to the
sequence.
The ratio of metallothionein in its native embodiment to heavy metals is 1:7.
In one embodiment, the ratio of metallothionein to a heavy metal, e.g.,
cisplatin by
weight is about 5:3. Any suitable ratio may be applied depending upon the
modified
MT selected, the heavy metal ion selected, and the number of binding sites for
the
heavy metal available on the modified MT. One of skill in the art may readily
select a
suitable ratio.
Incubation temperature can range from about 25 to about 42°C . In
one
embodiment, the temperature is about 37°C. Still other ranges may be
readily
selected by one of skill in the art. Similarly, the incubation time can range
from 60
minutes to 48 hours. In one embodiment, the incubation time is 48 hours. Still
other
ranges may be readily selected by one of skill in the art.
Any number of conventional buffers may be selected for this use depending
upon the functional requirements of the formulation as determined by one
skilled in
the art. See, e.g., buffers listed in Good, N. E. et al. (1966) Biochernistry
5, 467 and
Good, N. E., and Izawa, S. (I972) Methods Er7zyrnol. 24, 53 The buffer may be
selected from a variety of buffers known to those of skill in the art to be
used in the
compositions of the invention and include, without limitation, phosphate
buffered
saline (PBS) or isotonic saline, such as ISOTON II (US Patent 3,962,125), Tris
buffer,
the organic buffer N-(2-Acetamido)-2-iminodiacetic acid (ADA), or
pyrophosphate
buffer, acetate buffers, succinate buffers, maleate buffers, citrate buffers,
imidazole
buffers, carbonate buffers, MES buffer, MOPS buffer, and HEPES buffer, among
many that may be readily selected by one of s1ei11 in the art. In one
embodiment, the
buffer was IOmM HEPES buffer. Thereafter the resulting product is dialysed
against
another suitable buffer, e.g., PBS or carbonate buffer.
Alternative steps for preparation can include stripping all heavy metals from
the MT sequence by placing the sequence into solution in a suitable buffer,
e.g.,
l OmM HEPES buffered to pH 3.0, and degassing the solution with dry nitrogen
to
strip all heavy metals. A desired heavy metal, e.g., zinc, may be conjugated
to the
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WO 2005/072270 PCT/US2005/002023
MT sequence by dialysis against a buffer. One such buffer contains 2mM beta-
mercaptoethanol, and 1mM zinc chloride in IOmM HEPES pH 7.4
One of skill in the art may readily assemble the compositions and components
of this invention given the teachings provided herein, without resorting to
undue
experimentation.
D. Pharmaceutical Fof~nzulatiohs
Pharmaceutical compositions of the present invention, in one embodiment,
contain a modified MT sequence conjugated to molecules of a divalent metal ion
or
components thereof as described above in a pharmaceutically acceptable carrier
with
other optional suitable pharmaceutically inert or inactive ingredients. In
another
embodiment, pharmaceutical compositions of the present invention contain one
or
more compositions, components or conjugates described above or with one or
more
different therapeutically useful reagents. In one embodiment, a single
modified MT-
divalent metal ion complex is present in a single composition. In another
embodiment, two or more different modified MT-divalent metal ion complexes are
combined with one or more chemotherapeutic agents and/or biological agents,
radiological agents, and/or other therapeutic agents as described below.
The pharmaceutical compositions of this invention include the modified MT-
metal ion complexes of this invention formulated neat or with one or more
pharmaceutical carriers for administration, the proportion of which is
determined by
the solubility and chemical nature of the compound, chosen route of
administration
and standard pharmacological practice. The pharmaceutical carriers) may be
solid or
liquid. Formulations may incorporate both solid and liquid carriers.
A solid carrier can include one or more substances which may also act as
flavoring agents, lubricants, solubilizers, suspending agents, fillers,
glidants,
compression aids, binders or tablet-disintegrating agents; it can also be an
encapsulating material. In powders, the carrier is a finely divided solid
which is in
admixture with the finely divided active ingredient. In tablets, the active
ingredient is
mixed with a carrier having the necessary compression properties in suitable
proportions and compacted in the shape and size desired. Suitable solid
carriers
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WO 2005/072270 PCT/US2005/002023
include, for example, calcium phosphate, magnesium stearate, talc, sugars,
lactose,
dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl
cellulose,
polyvinylpyrrolidine, low melting waxes and ion exchange resins, crystalline
cellulose, binders such as hydroxypropyhnethyl cellulose, coating agents such
as
hydroxypropylmethyl cellulose and terephthalate thereof, lubricants such as
zinc
stearate and aluminum stearate.
Liquid carriers are used in preparing solutions, suspensions, emulsions,
syrups, elixers and pressurized compositions. The active ingredient can be
dissolved
or suspended in a pharmaceutically acceptable liquid carrier such as water, an
organic
solvent, a mixture of both or pharmaceutically acceptable oils or fats.
Carriers
include glycerol, propylene glycol, liquid polyethylene glycol, and the like.
The
liquid carrier can contain other suitable pharmaceutical additives such as
solubilizers,
emulsifiers, isotonic agents, sugars, sodium chloride, anti-oxidants, buffers,
bacteriostats, preservatives, sweeteners, flavoring agents, suspending agents,
thickening agents, surfactants, colors, viscosity regulators, stabilizers or
osmo-
regulators, tensio-active agents. Suitable examples of liquid carriers for
oral and
parenteral administration include water (partially containing additives as
above, e.g.
cellulose derivatives, preferably sodium carboxymethyl cellulose solution),
alcohols
(including monohydric alcohols and polyhydric alcohols, e.g. glycols) and
their
derivatives, lethicins, and oils (e.g. fractionated coconut oil and arachis
oil). For
parenteral administration, the carrier can also be an oily ester such as ethyl
oleate and
isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form
compositions for parenteral administration. The liquid carrier for pressurized
compositions can be a halogenated hydrocarbon or other pharmaceutically
acceptable
propellant, solubilizing and dispersing agents such as polyoxyethylene
hardened
castor oil, stabilizers, pH adjusters, and isotonicity-imparting agents,
preservatives,
anti-bacterial and anti-fungal agents, liposomes, mannose, glucose and
balanced salt
solutions, phosphate buffered saline, diethyl ether, isopropyl ether,
halothane, or
trifluorotrichloroethane.
Additionally, the compositions of the present invention may be formulated
alone in a composition of the invention or may be formulated in combination
with
27
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WO 2005/072270 PCT/US2005/002023
other compounds of the invention and other compounds known in the art of
cancer
treatment. Compounds of the present invention may also be used in combination
with
other therapeutic agents. In certain embodiments, such other such agents
include, but
are not limited to chemotherapeutic agents, such as anti-metabolites, DNA
damaging,
microtubule destabilizing, microtubule stabilizing, actin depolymerizing,
growth
inhibiting, topoisomerase inhibiting, HMG-CoA inhibiting, purine inhibiting,
pyrirnidine inhibiting, metaloproteinase inhibiting, CDI~ inhibiting, caspase
inhibiting, proteaosome inhibiting, angiogenesis inhibiting, differentiation
inducing
and immunotherapeutic drugs, and compositions for promotion of TGF-(3 response
and/or apoptosis. These agents include, but are not limited to, anthracycline
antibiotics such as doxorubicin and mitoxantrone, estramustine, vinblastine,
paclitaxel, etoposide, cyclophosphamide, cisplatin, carboplatin, adriamycin, 5-
fluorouracil, camptothecin, actinomycin-D, mitomycin C, adriamycin, verapamil,
podophyllotoxin, and the lilee (with or without the addition of steroid
drugs); anti-
androgens (such as flutamide, bicalutamide, nilutamide, megestrol acetate,
adrenocorticotropic hormone secretion inhibitors, lcetoconazole, estrogens,
anti-
estrogens and LHRH production suppressors), immunomodulatory agents (including
cytolcines, chemokines, interferons, interleukins), a non-progestin/non-
estrogen
apoptosis promoting agent selected from the group consisting of the retinoids
(retinoic
acid, N-(4-hydroxyphenyl) retinamide-O-glucuronide, N-(4-hydroxyphenyl)
retinamide, O-glucuronide conjugates of retinoids, N-(4-hydroxyphenyl)
retinamide
and its glucuronide derivative, retinyl-[3-glucuronide, the glucuronide
conjugates of
retinoic acid and retinal, tretinoin, etretinate, arotinoid, isotretinoin,
retinyl acetate,
acitretin, adapalene, and tazarotene), adamantyl or adamantyl group
derivatives
containing retinoid-related compounds (e.g., 6-[3-(1-adamantyl)-4-
methoxyphenyl]-2-
naphthoic acid, 2-[3-(1-adamantyl)-4-methoxyphenyl]-5-benzimidazole carboxylic
acid, and 6-[3-(1-adamantyl)-4,5-methylenedioxyphenyl]-2-naphthoic acid; 2-
methoxyestradiol), progestin, 1-O-acetylbritannilactone, 1,6-O,O-
diacetylbritannilactone, a 2-nitroimidazole derivative (e.g. 1-(2,3-dihydroxy-
1-
(hydroxymethyl)-propoxymethyl)-2-nitroimidazole, 1-(4-hydroxy-2-
butenyloxymethyl)-2-nitroimidazole and 1-(2,3-dihydroxypropoxymethyl)-2-
28
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nitroimidazole), benzamide riboside, synthetic glycoamines, TNF-a, anti-Fas
antibody, thapsigargin, TGF-(3 (TGF-(3-1, TGF-(3 -2 or TGF-(3-3), non-
progestin/non-
estrogen TGF-~3 inducing agents, polyclonal antibodies, monoclonal antibodies,
dietary flavanoids, anti-inflammatory drugs, monoterpenes, S-adenosyl-L-
methionine,
selenium, and vitamin D compound.
Other chemotherapeutic agents include biological agents such as a p53 protein
or gene, Mycobacterium phlei (M. Phlei) DNA (M-DNA) & DNA complexed with
M. phlei cell wall (MCC), extract of Melothria indica Lou, fetuin, Apogen P-
la,
Apogen P-lb, Apogen P-lc, Apogen P-2, and Apogen L. Further apoptotic agents
include radiological apoptotic agents such as radioisotopes and DNA damaging
radiation such as X-rays, UV-light, gamma-rays and microwaves.
Other therapeutic agents not listed above, but which are beneficial in
combination therapies of the invention, are contemplated as within the
invention.
In one embodiment, compositions of the invention and/or other agents may be
administered in a single composition. However, the present invention is not so
limited. In other embodiments, compositions of the present invention may be
administered in one or more separate formulations from other compositions of
the
invention, chemotherapeutic apoptotic agents, biological apoptotic agents,
radiological apoptotic agents, or other agents as is desired.
Compounds and compositions of the present invention may be formulated for
administration via sterile aqueous solution or dispersion, aqueous suspension,
oil
emulsion, water in oil emulsion, site-specific emulsion, long-residence
emulsion,
sticky-emulsion, microemulsion, nanoemulsion, liposomes, microparticles,
microspheres, nanospheres, nanoparticles, minipumps, and with various natural
or
synthetic polymers that allow for sustained release. The compounds of the
present
invention may also be formulated into aerosols, tablets, pills, sterile
powders,
suppositories, lotions, creams, ointments, pastes, gels, hydrogels, sustained-
delivery
devices, or other formulations used in drug delivery.
The particular formulation or formulations used will vary according to the
routes) of administration desired. For example, injectable formulations can be
prepared by combining the compositions with a liquid. The liquid can be
selected
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WO 2005/072270 PCT/US2005/002023
from among water, glycerol, ethanol, glycols, such as propylene glycol and
polyethylene glycol, oils, and mixtures thereof, and more preferably the
liquid carrier
is water. In one embodiment, the oil is vegetable oil. Optionally, the liquid
carrier
contains a suspending agent. In another embodiment, the liquid carrier is an
isotonic
medium and contains about 0.05% to about 5% suspending agent.
In one embodiment, the pharmaceutical composition is in unit dosage form,
e.g. as tablets or capsules. In such form, the composition is sub-divided in
unit dose
containing appropriate quantities of the active ingredient; the unit dosage
forms can
be packaged compositions, for example, packaged powders, vials, ampoules, pre-
filled syringes or sachets containing liquids. The unit dosage form can be,
for
example, a capsule or tablet itself, or it can be an appropriate number of any
such
compositions in package form.
D. Plaarmaceuticall~its
The present invention provides kits or packages of pharmaceutical
formulations including the compounds or compositions described herein. The
kits are
also preferably organized to indicate a single oral or intravenous formulation
or
combination of oral formulations to be taken at each desired time, preferably
including oral tablets to be taken at each of the times specified, and more
preferably
one oral tablet will contain each of the combined periodic dosages indicated.
The left can also include one or more chemotherapeutic agents, biological
apoptotic agents, or other therapeutic agents, such as one or more agents)
selected
from among those previously described. One of skill in the art would readily
be able
to formulate a suitable amount of the above-described agents for use in the
kits of the
invention. Kits containing radiological agents in combination with the
compositions
of the invention are also contemplated.
When the compounds or compositions described herein are to be delivered
continuously, a package or lcit can include the compound in each dosage unit
(e.g.
solution, lotion, tablet, pill, or other unit described above or utilized in
drug delivery).
When the compound is to be delivered with periodic discontinuation, a package
or kit
can include placebos during periods when the compound is not delivered. When
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varying concentrations of a composition, of components of a composition, or of
relative ratios of compounds or agents within a composition over time is
desired, a
package or kit may contain a sequence of dosage units, so varying.
A number of packages or kits are known in the art for the use in dispensing
pharmaceutical agents for such use. Preferable, the package has indicators for
each
period, and more preferably is a labeled blister package, dial dispenser
package, or
bottle. The packaging means of a lcit may itself be geared for administration,
such as
an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from
which the
formulation may be applied to an infected area of the body, such as the lungs,
injected
into a subject, or even applied to and mixed with the other components of the
kit.
The compositions of these kits also may be provided in dried or lyophilized
forms. When reagents or components are provided as a dried form,
reconstitution
generally is by the addition of a suitable solvent. It is envisioned that the
solvent also
may be provided in another packaging means.
The lcits of the present invention also will typically include a means for
containing the vials in close confinement for commercial sale such as, e.g.,
injection
or blow-molded plastic containers into which the desired vials are retained.
Irrespective of the number or type of packages, the kits of the invention also
may
include, or be packaged with a separate instrument for assisting with the
injection/administration or placement of the ultimate complex composition
within the
body of an animal. Such an instrument may be an inhalant, syringe, pipette,
forceps,
measuring spoon, eye dropper or any such medically approved delivery means.
Other
instrumentation includes devices that permit the reading or monitoring of
reactions in
vitro.
In one embodiment, a pharmaceutical kit of the invention including at least
one composition according to the invention in a dosage unit. In still other
embodiments, pharmaceutical kits of the invention also contain
chemotherapeutic
apoptotic agents, biological apoptotic agents, and/or other therapeutic agents
as
described above. In still other embodiments, pharmaceutical kits of the
invention
employ the above-described compositions along with radiological agents and
treatments.
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E. lVlethods of Treat~zent
The modified MT-heavy metal complexes of this invention, components
thereof and pharmaceutical composition containing same are useful in methods
for
treating cancer. Specific cancers to be treated with the compositions of the
invention
include estrogen negative breast cancer, estrogen positive breast cancer,
prostate
cancers (including androgen-independent prostate cancer), ovarian cancer,
bladder
cancer, brain cancer, head and neck cancer, kidney cancer, lung cancers such
as small
cell lung cancer and non-small cell lung cancer, myeloma, neuroblastoma/
glioblastoma, pancreatic cancer, skin cancers, liver cancers, melanoma, colon
cancer,
cervical carcinoma, and leukemia, retinoblastoma, pancreatic islet carcinoma,
or
other epithelial-derived cancers. The methods of this invention have
particular
applicability to those cancers which are currently treated with divalent heavy
metal
ions. Among such cancers are included, without limitation, brain cancer, head
and
neck cancers, thyroid cancer, lung cancer, ovarian cancer, prostate cancer,
and bladder
cancer.
Such methods include administering to a mammalian subject, preferably a
human subject an effective amount of the modified MT-heavy metal complexes of
this invention. Such treatment permits delivery of suitable dosages of
divalent metal
ions to treat the cancer, and yet inhibits the renal uptake of the heavy
metals. Thus
such methods have an advantage over currently employed chemotherapeutic
treatments with heavy metals and are likely to permit the use of current doses
more
safely or higher, more effective dosages with less danger of toxicity. For
example,
current dosages of cisplatin for cancer therapy are between 50 to 270 mg/m2.
Because
the compositions of this invention do not accumulate in the kidneys, higher
doses of
the heavy metals, which are more likely to be able to kill a targeted tumor or
cancer
cell, may be administered with less concern for toxicity to the treated
patient.
Methods employing these compositions inhibit renal uptake of the therapeutic
divalent metal ions by megalin receptors in the proximal tubules of the
kidneys.
Thus, these methods also permit current dosages of heavy metals to be employed
in
anti-cancer therapies more safely.
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In one embodiment, a method of treating or preventing the development of
cancer in a mammalian subject comprising treating cancer cells of said subject
with a
composition of the invention is contemplated, either ifa vivo or ex vivo. The
pharmaceutical compositions of the present invention may be administered to a
subject via one or more routes to contact the cancer cells, as desired. For
example,
the compositions may be administered via oral, topical, systemic, enteral,
parenteral
(e.g., formulated for injection via the intravenous, intramuscular,
subcutaneous,
intracutaneous, or even intraperitoneal routes (e.g. by drip infusion)),
subcutaneous,
intra-portal, intra-prostatic, intra-muscular, intra-venous, intra-arterial,
intra-dermal,
intra-thecal, intra-lesional, intra-tumoral, intra-bladder, intra-vaginal,
intra-ocular,
intra-rectal, intra-pulmonary, intra-spinal, transdermal, and subdermal
routes.
Further, the compositions may be delivered via placement within cavities of
the body,
regional perfusion at the site of a tumor or other desired location, nasal
inhalation,
pulmonary inhalation, impression into skin and electrocorporation. The routes)
of
administration will vary according to the cell(s), tissue(s), organ(s), or
systems) to be
treated.
In a further embodiment, the compounds are delivered transdermally or by
sustained release through the use of a transdermal patch containing the
composition
and an optional carrier that is inert to the compound, is nontoxic to the
skin, and
allows for delivery of the compound for systemic absorption into the blood
stream.
Such a carrier can be a cream, ointment, paste, gel, or occlusive device. The
creams
and ointments can be viscous liquid or semisolid emulsions. Pastes include
absorptive powders dispersed in petroleum or hydrophilic petroleum. Further, a
variety of occlusive devices can be utilized to release the active reagents
into the
blood stream and include semi-permeable membranes covering a reservoir contain
the
active reagents, or a matrix containing the reactive reagents.
The use of sustained delivery devices can be desirable, in order to avoid the
necessity for the patient to take medications on a daily basis. The term
"sustained
delivery" is used herein to refer to delaying the release of an active agent,
i.e., a
compound of the invention, until after placement in a delivery environment,
followed
by a sustained release of the agent at a later time. A number of sustained
delivery
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WO 2005/072270 PCT/US2005/002023
devices are known in the art and include hydrogels (US Patent Nos. 5,266,325;
4,959,217; 5,292,515), osmotic pumps (US Patent Nos. 4,295,987 and 5,273,752
and
European Patent No. 314,206, among others); hydrophobic membrane materials,
such
as ethylenemethacrylate (EMA) and ethylenevinylacetate (EVA); bioresorbable
polymer systems (International Patent Publication No. WO 98/44964 and US
Patent
Nos. 5,756,127 and 5,854,388); and other bioresorbable implant devices
composed of,
for example, polyesters, polyanhydrides, or lactic acid/glycolic acid
copolymers (US
Patent No. 5,817,343). For use in such sustained delivery devices, the
compounds of
the invention can be formulated as described herein. See, US Patent Nos.
3,845,770;
3,916,899; 3,536,809; 3,598,123; and 4,008,719.
The methods of this invention involve administering such compositions in
effective amounts to induce apoptosis in the cancer cells, while minimizing
adverse
impacts on non-cancer cells of the patient. Dosages of the compounds and
compositions of the present invention vary with the particular compositions
employed, the route of administration, the severity of the symptoms presented,
the
particular subject being treated, and the subjects other medications and
treatment, as
well as the subject's medical history. Precise dosages for intravenous, oral,
parenteral, nasal, or intrabronchial administration can be determined by the
administering physician based on experience with the individual subject
treated. An
effective therapeutic dosage will contain a dosage sufficient to induce
apoptosis of
cancer cells.
The amount of the compound of the invention present in each effective dose is
selected with regard to consideration to the half life of the compound, the
identity
and/or stage of the cancer, the patient's age, weight, sex, general physical
condition
and the like. The amount of active component or compound required to induce an
effective apoptotic effect on cancer cells without significant adverse side
effects
varies depending upon the pharmaceutical composition employed and the optional
presence of other components. Suitable dosages of compositions used to treat
cancers
as described herein can range from 1.0 pg to 500 mg MT sequence(s)/Icg patient
body
weight. In one embodiment, the dosage is at least 10 p,g/kg. In another
embodiment,
the dosage is at least 100 pg/lcg. In another embodiment, the dosage is at
least 500
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WO 2005/072270 PCT/US2005/002023
pg/kg. In another embodiment, the dosage is at least 1 mg/kg. In another
embodiment, the dosage is at least 10 mg/kg. In another embodiment, the dosage
is at
least 50 mg/lcg. In another embodiment, the dosage is at least 100 mg/kg. In
another
embodiment, the dosage is at least 250 mg/leg. In another embodiment, the
dosage is
at least 400 mg/kg. In another embodiment, the dosage is at least 500 mg/kg.
In
another embodiment, each dose will contain between about 5 pg peptide/kg
patient
body weight to about 10 mg/kg. Generally, a useful therapeutic dosage is
between 1
to 5 mg peptide/kg body weight. Another embodiment of a useful dosage may be
about 500 pg/kg of peptide. Other dosage ranges may also be contemplated by
one of
skill in the art. For example, dosages of the peptides of this invention may
be similar
to the dosages discussed for other cancer therapeutics. As one embodiment, the
dosages may be provided in terms of mg heavy metal per meter square. For
example,
the amount of the MT-heavy metal complex to be delivered may be greater than
50
mg/m2 and possibly greater than 100mg/m2 or greater than 270 mg/m2. Initial
doses
of a composition of this invention may be optionally followed by repeated
administration for a duration selected by the attending physician. Dosage
frequency
may also depend upon the factors identified above, and may range from 1 to 6
doses
per day for a duration of about 3 days to a maximum of no more than about 1
week.
The compositions of this invention may also be administered as a continuous
infusion
for about 3-5 days, the specific dosage of the infusion depending upon the
half life of
the compound. The compounds of this invention may also be incorporated into
chemotherapy protocols, involving repetitive cycles of dosing. Selection of
the
appropriate dosing method would be made by the attending physician.
In another embodiment of this invention, the method of treating or preventing
the development of cancer in a mammalian subject involves exposing the subject
to
one or multiple (e.g. 2, 3, 4, or more) chemotherapeutic apoptotic agents,
biological
apoptotic agents, radiological apoptotic agents, or other therapeutic agents
described
herein. Such combination treatment may occur by administering compositions
containing multiple active ingredients, as described above. However, this
invention
also encompasses a method of administration of anti-cancer agents or therapies
in
conjunction with a composition containing a modified MT-heavy metal ion
complex
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WO 2005/072270 PCT/US2005/002023
as described above. In one embodiment, a different chemotherapeutic agent is
administered before treatment with a composition of the invention. In another
embodiment, a different chemotherapeutic agent is administered after treatment
with a
composition of the invention. In still another embodiment, a chemotherapeutic
or
biological apoptotic agent is administered before, during or after treatment
with a
composition of the invention.
In still another embodiment, the patient is exposed to radiological treatment
administered before, during or after treatment with a composition of the
invention.
Where a radiological agent is desired in combination with one or more of the
compounds or compositions of the present invention, dosage may be determined
by
an administering physician according to standard regimens taking into account
other
factors including other treatments applied in combination. For example, the
appropriate regimen of radiation dosage ranges for X-rays ranges from daily
doses of
50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single
doses of
2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and
depend on
the half life of the isotope, the strength and type of radiation emitted, and
the uptake
by cells.
The compositions of this invention may also be employed in a method for
treating heavy metal poisoning in a mammal, preferably a human. A modified MT
sequence or fragment as described above, e.g., the hinge region sequence
SCICKSCC
itself, is administered to the subject in an amount sufficient to compete i~
vivo with
naturally circulating MT for complexation with any heavy metal in the
circulation.
Excess amounts of the modified MT may be employed to scavenge any heavy metal
in the circulation. The modified MT-heavy metal complexes are then eliminated
from
the body by bypassing the megalin receptors in the kidneys. Suitable dosages
of the
modified MT sequences can be determined by an attending physician based upon
the
condition of the patient, the heavy metal involved, the level of poisoning,
etc.
The identification of megalin as the receptor for MT binding in the renal
tubules also permits a method for the treatment of heavy metal poisoning by
treating a
patient known to have ingested or to have been exposed to heavy metals with an
excess amount of a known megalin ligand. Many such ligands are known in the
art,
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WO 2005/072270 PCT/US2005/002023
as evidenced by the publications cited herein, as is the hinge region itself.
By treating
the patient with an excess of a suitable megalin ligand, particularly a ligand
that binds
megalin with an avidity greater than that of naturally-occuring MT, megalin
receptors
in the kidneys can be bound by such ligands, thereby reducing the number of
receptors available to bind the circulating MT that forms complexes with the
heavy
metal in the patient's circulatory system. In this manner, the naturally-
formed
complexes can be excreted without opportunity to bind in the renal tubules.
Thus, a
patient so treated, may eliminate more of the heavy metal and experience less
toxicity.
Suitable dosages of the megalin ligands can be determined by an attending
physician
based upon the condition of the patient, the heavy metal involved, the level
of
poisoning, etc. However, such dosages are likely to be quite high, perhaps
over 100
times the amount of naturally-occurring MT.
F. Method fof~ Developing Novel Therapeutics fos~ Treatme~~t of Caficer~ or
1 S Fleavy Metal Poisoning.
The present invention and the discovery of the megalin receptor of MT
provides yet another aspect of this invention, i.e., a method for identifying
a test
compound for cancer treatment. For example, a battery of new or known
compounds
is assayed for the ability to compete with naturally-occurring MT for binding
to the
megalin receptor. Compounds that bind megalin more avidly than MT may be
useful
for the treatment of heavy metal poisoning, as described above. In contrast,
compounds that bind megalin less avidly than MT and can be engineered to also
bind
therapeutic metal ions may be useful as cancer therapeutics, in the same
manner as
described above for the modified MT sequences.
Thus, in one embodiment, a method for identifying therapeutic compounds
includes contacting in vitro a sample of immobilized megalin receptor with a
test
compound and naturally-occurring MT. Once any unbound substances are
eliminated, the relative amounts of MT and the test compound which are
immobilized
on the megalin receptor are measured. The presence of excess test compound
indicates a compound that may be useful for the treatment of heavy metal
poisoning.
An excess of bound MT indicates that the test compound binds rnegalin less
avidly
37
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than MT and, if the test compound is also capable, or may be made capable of
binding
heavy metal ions, the test compound may be useful to deliver such ions ifZ
vivo for
cancer treatment.
Other assay protocols known to the art may be designed which employ
binding to megalin as the critical determination for identification of a
suitable
compound for use as a drug for this purpose.
G. Exa~zples
The following examples illustrate various aspects of this invention. Examples
1-6 provide evidence indicating that megalin binds MT and is the receptor
responsible
for the uptake of Gd-MT in the proximal convoluted tubules. Megalin is by far
the
most quantitatively important mechanism of MT uptake into the renal proximal
tubule. First, SPR directly demonstrates binding of the purified proteins in a
dose-,
ion-, and pH-dependent manner. Second, antibody interference experiments show
that >90% of the MT binding on brush-border membrane vesicles, and cellular
uptake
into BN-16 cells, can be displaced or inhibited specifically with anti-
megalin, but not
control, antisera. Finally, megalin and MT colocalize at the cellular level in
$uorescent microscopy studies. Megalin and MT colocalize and internalize con-
comitantly before separating in the late endosomal pathway. The Examples 7 - 8
demonstrate that a reagent comprising a mutant MT that does not bind megalin,
but
binds heavy metals, is taken up by cancer cells and maintains clinical
efficacy.
These examples do not limit the scope of this invention which is defined by
the appended claims. One skilled in the art will appreciate that although
specific
reagents and conditions are outlined in the following examples, modifications
can be
made which are intended to be encompassed by the spirit and scope of the
invention.
EXAMPLE 1: ANIMALS, REAGENTS, ANTIBODIES, STATTS~hICS
Male Sprague-Dawley rats (200-250 g) were obtained .from Sasco (Omaha,
NE). All reagents were from Sigma (St. Louis, MO) unless otherwise stated.
These studies used commercially available MT-I, isolated from either rabbit
liver or horse kidney, which is a highly conserved mammalian isoform. All
known
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class I sequences contain 61 or 62 amino acids with 20 conserved cysteine
residues
and are able to bind up to 7 equivalents of divalent metal ions, commonly a
mixture of
zinc and cadmium. Although Zn-MT and Cd-MT differ dramatically in their toxic
effects, they produce virtually identical profiles in their binding and uptake
(Dorian C
and Klaassen CD. 1995 Fundana Appl Toxicol 26: 99-106). In solution, MT tends
to
form oligomers (Tang W et al, 1999 J Anal Toxicol 23: 153-158). Therefore, MT
was
used as received, rather than saturated with cadmium in an extra step, to
maximize the
structural integrity of the metalloprotein during our analyses. The inclusion
of
recombinantly expressed MT samples in these examples is an important control
for
any impurities in the commercial reagents. The supplier-reported metal assays
of MT
samples show ~7% metals by mass, which indicated complete occupation of all
metal-
binding sites by zinc and/or cadmium.
In preliminary experiments, the inventors found that DTT, which is frequently
used as a reducing agent in solution with MT to prevent oxidation of MT,
potently
denatures megalin, abolishing the binding of knawn megalin ligands. Thus the
inventors continually used an internal control having cysteine content similar
to active
peptides to correct for the nonspecific effects of suifydryl binding. The
ability of the
MT-fragment peptide 2 (aa 10-26 of SBQ ID NO: I ) partially to inhibit binding
of
1V1'C to brush-border membranes, but not uptake in BN-16 cells, falls into
this
category.
Purified human megalin and cubilin receptors were obtained by detergent
solubi lization of renal cortex brush-border membranes followed by affinity
chromatography using immobilized receptor-associated protein (Moestrup SK. et
al,
1998 .Jf3iol Chern 273: 5235- 5242). Polyclonal antibodies against cubilin,
megalin,
and transferrin were raised against proteins purified by immunoaf~lnity
chromatography using previously reported monoclonal antibodies coupled to
Sepharose 4B (Hannnond TG et al, 1994 Arn .l Pl~ysiol Renal Flr.riel
Electrolyte
Phy.siol 267: F516-F527; Moestrup, cited above, Sahali D et al, 1988 JExp
tiled 167:
213-218; Sahali D et al, 1993 ,~Inr .JPatlrol 142: 1654-1667). These
antibodies were
monospecific by Im11111110b101 111g on whole brush-border preparations and by
immunoprecipitation of biosynthetically labeled yolk sac epithelial cells in
culture.
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Among the antibodies employed were: anti-neurolcinin-1 (NK1)/substance-P
receptor
antiserum (Dr. Jacques Gouraud, Gif sur-Yvette, France) (Bret-Dibat JL, et
al_, 1994
,INct.ct~oclzenz 63: 333-343); anti-giantin (Dr. H. P. Hauri, University
ofBasel>.
Antibodies to angiotensin II type 1 (ATI) receptor (Santa Cruz Biotechnology,
Santa
S CI'L1Z, CA).; fluorescent secondary antibodies: mouse FITC-anti-goat
antibod~~ (Dako,
Carpinteria, CA) or goat anti-mouse, goat anti-rabbit, and donkey anti-sheep
antibodies, all conjugated to Alexa 488 (Molecular Probes, Eugene, OR).
Synthetic peptides corresponding to portions ofthe MT sequence (FIG. 1 )
were obtained from Biosource International, Carnarillo, CA). Recombinant
production of full-length MT (rMT fI) and the two individual domains (rMT- cx
and
rMT-~) were prepared as described in Atrian S, et al., "Recombinant synthesis
and
metal-binding abilities of mouse metallothionein l and its a-and j3-domains."
In:
Metalle~tlziorzei>z Ilr, ed. Klaassen CD. Basel: Birkha"user Verlag, 1999, p.
55-61. The
protein yields and atomic absorption validation of metal content of the
recombinant
proteins are shown in Table 1.
TABLE 1: Protein yield and heavy metal content of recombinant mouse MT
proteins
Clone Protein Concentration/ Zn Content Predicted
as
Total Yield by AZx~ Zn/Protein Ideal
Ratio
by inductivelyZn/Protein
cou led plasmaRatio
rMT tl 1.18x10-' M12. l4 mg 6.73 7
total
rMT-a 1.37xI0~~ M/1.58 mg 3.93 4
total
rMT-~3 1.87x10''' M/2.01 mg 3.01 3
total
Data in the following exayples are expressed as means ~ SE throughout the
manuscript. Statistical analysis was performed by analysis of variance and
Bonferroni
or Scheffe"s post hoc comparison. Flow cytometry data were also analyzed by
Kolgomorov-Srnirilov summation statistics (Young 1T. 1977 .I Histe~cJzen?
C'ytoc°hezrz
25: 935-94 I ).
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EXAMPLE 2: MOLECULAR STUDIES Or RECEPTOR-MT BINDING USING
S PR.
Cubilin and megalin were studied separately by using SPR, immobilizing
purified membrane-free samples of each receptor, and studying its interaction
with
rabbit liver MT. The interaction of MT with megalin was assayed with a BIACORE
3000 biosensor system (Biacore). In surface plasmon resonance (SfR), one
protein is
immobilized to a dextran-coated gold surface. Injection of a soluble protein
produces
a signal change that is directly proportional to the mass of bound protein and
is
reported as resonance units (RU). Megalin (0.025 mg/ml in 10 mM acetate, pH
4.53)
was immobilized (1,000-3,000 RU) in one flow cell on a CM5 biosensor chip
using
standard primary amine-coupling methods as detailed by the manufacturer. An
equal
amount of either ovalbumin or casein was immobilized in a second flow cell to
provide real-time reference correction for instrumental aaifacts and
nonspecific
binding events.
Rabbit liver MT was injected over both flow cells at room temperature in
HOPES-buffered saline (HBS), pH 7.4, containing 2 mM Ca, 2 mM Mg, and 0.005%
surfactant P20. Maximum reproducibility was obtained when
0.0008°f° sodium
dextran sulfate (catalog no. 17-0340-O1, Pharmacia Biotech) was also included
in the
buffer. Equilibrium dissociation constants (ICd) were determined from steady-
state
binding measurements at concentrations of 75, 150, 300, 600, 1,200, and 2,400
pghnl
MT. Proteins were typically injected at flow rates of 50 plhnin for 3 min and
then
allowed to dissociate for 5 min.
Because MT is a low-affinity 1 igand, no regeneration (removal of bound
protein by injection ofa second, typically harsh, solvent) was necessary. The
"double-referencing" technique (Myszka, cited above) was used to eliminate
additional instrumental artifacts. The blank injections used for this
procedure were
identical to sample solutions except for the omission of MT. Thermodynamic
constants were calculated using Biacore's BIAevaluation 3.1 software.
The dose-dependent binding to megalin (not shown) uniformly increased with
dose over a 32=fold increase in concentration, 75-2,400 ~g/ml. The observed
variations and noise were normal for the very low signal levels used. The fit
of
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maximum responses obtained after 2.5 min. is illustrated in the graph of F IG.
2. Even
ai high MT concentrations, >90% saturation was not achieved, and therefore
some
errors occurred in the fit. An approximate fit using the maximum (but non
equilibrium)
responses obtained at each concentration yielded an estimated dissociation
constant of
-s
9.$ x IO M (FIG. 2). Repeated experiments consistently indicate the binding of
~0.7-0.9 mol of MT/mol of megalin, consistent with ane binding site. In
contrast, no
binding of MT to cubilin was observed.
The binding shown in FLG. 2 was specific for megalin and depended on metal
ions but not an the MT source. Omitting either Ca or Mg from the sample
buffers
abolished the binding (data not shown); both appeared to be required. Samples
of MT
from horse kidney and from rabbit liver provided nearly identical results
(data not
shown). Interestingly, oligomerized MT bound mare effectively to megalin than
did
the monomer. Nondenaturing gel electrophoresis showed that over time, MT forms
trimers, tetramers, and even much larger oligomers (data not shown). The
binding of
such molecules to megalin was significantly stronger. Owing to dii~culti~s in
purifying these aligomers, the actual binding constants for oligomers could
not be
determined with any precision. Qualitatively, compared with manomeric MT,
oligomeric MT dissociated much more slowly, and harsher conditions were
required
to dislodge it from immobilized megalin. Using the tetramer as a basis for
calculations, one may estimate a 100-fold change in Kd (7 X 10-~ M).
EXAMPLE 3: INI~LBITION OF MT B1NDLNG BY MEGALIN L:IGANDS
AND SMALL PEPTIDES DERIVED F ROM MT.
A. Peptides
A series of peptides spanning the sequence of rabbit liver MT were
prepred and used SPR to study their effect on the binding of MT to megal in
(see FIG.
1). Six 16-amino acid peptides, spanning the entire MT sequence with each
overlapping its neighbors by 7 amino acids (Biosource International; I<agi JHR
and
Vasak M., "Chemistry of mammalian metallothionein." In: Metallothionein in
Biology afid Medicif7e, eds. Klaassen CD and Suzuki KT., Boca Raton, FL: CRC,
1991, p. 49-60) were used to inhibit the binding of MT to megalin. The
SCKI~SCC
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peptide (aa 28-34 of SEQ ID NO: 1)represented the overlap sequence between two
of
these peptides (Biosource International). The peptide concentrations were ~5
pM,
while rabbit liver MT was 250 pg/ml, corresponding to the ligand Kd
(estimated).
B. PI"2~C11'atl~1'2 Uf YGCOT'19~i)9aYll rraot~.re MT tn~d a- ahd ~3-sacbunit
Recombinant fragments of mouse MT were produced successfi~lly and
reproducibly (Atrian et al., cited above) by making recombinant I~T subunits
in
Esehef~ichia coli using a GST fusion vector followed by thrombin cleavage to
release
the free MT subunit. This approach is more practical than site-directed
mutagenesis,
as most attempts to produce recombinant MT have been characterized by very low
yields or by mixtures of several short cleavage fragments of the MT molecule
(Huang
PC et al, ''Native and engineered metallothioneins." In: Metallothionein. ij~
Biolo4~
crjzd Medicine, ed. I~laassen CD and Suzuki KT., Boca Raton, rL: CRC, 1991, p.
87-
101). The thrombin cleavage leaves three amino acids, speciftcally SCM.
derived
from the COOL-I terminus of the GST, on the NfI~ terminus of the product.
To understand the data, the inventors postulated the critical binding site on
MT to be the intradomain SCI<KSCC (aa 28-34 of SEQ ID NO: 1) region, with SCIC
representing the COON-terminal end of the (i-subunit and KSCC the NHS-terminal
start of the a-subunit. The recombinant a-subunit, therefore, has a
conservative GST-
derived SCM substitution for SCK on its NIh terminus, leaving the postulated
critical
SCKLCSCC sequence (aa 28-34 of SEQ ID NO: 1) essentially intact. The
recombinant
(3-subunit starts with SCM-and ends in SCK, rendering the postulated critical
SCICKSCC (aa 28-34 of SEQ 1D NO: 1) disrupted. The full-length recombinant MT
has an intact SCKKSCC (aa 28-34 of SEQ ID NO: 1) sequence as well as an
additional NHZ-terminal SCM. Atomic absorption (inductively coupled plasma)
analysis of the zinc content of the recombinant subunits proved them to be at
the
predicted heavy metal content to within the error of the methods (see Table 1
).
C. 'SPR analysis of binding.
Megalin was immobilized as described above in Example 2. An equal amount
of transferrin (0.10 ~.g/ml in 10 mM acetate, pH 4.96) was immobilized in a
second
flow cell to provide real-time reference correction. Dose-dependent peptide
binding
was examined by injecting the peptide at concentrations ranging from 0 to 500
pg/ml.
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Inhibition of MT binding by peptide was examined by injecting rabbit liver MT
as
described above and comparing the results to samples that contained varying
concentrations of peptide but were otherwise identical. No regeneration was
necessary. Additional artifacts were eliminated before curve fitting by
applying
double-referencing techniques. The blank injections used for this procedure
were
identical to sample solutions except for the omission of MT and peptide.
Peptides unrelated to MT but having a central ICK motif, specifically a v-
ATPase (I-subunit peptide with a KK motif (CLQKFEKKINQSPYEKR; SEQ ID NO:
5) and an apolipoprotein A-I peptide with KK motif (ALEEYTKKLNTQ; SEQ 1D
NO: 6; Biosource International), served as control peptides.
Protein concentrations were assayed by the Bradford method (Pierce
Biotechnology, Rockford, IL). Recombinant mouse MT proteins, and native mouse
MT as a control, were dialyzed into SPR binding buffer with magnesium and
calcium
under acidic conditions to remove the zinc. Aliquots of the proteins were
recon-
stituted with zinc by simple allcalinization in the presence of the metal, and
excess
metal was removed with resin. The proteins were used for SPR analysis or
redialyzed
into appropriate buffers for cell uptake studies.
Results obtained by injecting synthetic peptides in HEPES-buffered saline
(f IBS) containing 2 mM Ca and Mg in these initial qualitative SPR studies are
summarized in Table 2.
TABLE 2: Binding to megalin by peptides derived from MT and interference
with binding of the native protein using SPR techniques
Peptide Sequence Binding Competition
to with
Fragments Megalin MT for Megalin
Bindin
1 (SEQ ID KMDPNCSCATGNSCTCA No No
NO: 7)
2 (aal0-25 GNSCTCASSCKCKECK No No
of SEQ
ID NO: 1 )
3 (aal9-34 CKCICECKCTSCICKSCC Yes Yes
of SEQ
ID NO: 1 )
4 (aa2$-43 SC(~KSCCSCCPAGCTK Yes Yes
of SEQ
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ID NO: I )
(aa37-52 CPAGCTICCAQGCICKG Insoluble;l:nsoluble;
of SEQ no data
ID NO: 1.) no data
6 (aa44-61 CAQGCICKGASDKCSCCA No No
of SEQ
ID NO: 1 )
When using SPR, no reproducible inhibition of MT binding to megalin by
known megalin ligands was observed, but inhibition by some of the synthetic
peptides
corresponding to sequences within MT was noted. Commercial sources of (32-
5 microglobulimdissociated only with difficulty from the immobilized megalin,
leading
to erratic, nonreproducible binding, and loss of binding of control ligands
after the
harsh regeneration modalities necessary. For this reason, SPR assessment of
competitive X32-microglobulin binding with MT was impractical.
Interestingly, peptides 3 and 4 bound quite tightly to megalin and also
disrupted the binding of MT. Although peptides 1, 2, and 6 contain cysteines,
they
did not bird megalin, suggesting that the binding of peptides 3 and 4 is a
specific
interaction, rather than a nonspecific disulfide interaction between the
peptides and
megalin. Technical issues prevented direct confirmation; reduction with DT T
denatured megalin and abolished the binding of all ligands. Because the
behavior of
peptides 3 and 4 differed significantly from that of the other soluble
peptides,
attention focused on the overlap sequence these peptides have in common.
SPR analysis ofthe binding of hinge peptide SCK1CSCC (aa 28-34 of SEQ ID
NO: 1 ) in HBS containing 2 mM Ca and Mg and altered binding of MT to megalin
in
the presence of the peptide was assayed using SPR techniques of Example 2. In
the
resulting graph (not shown) traces represented the responses obtained with 63,
125,
250, and 500 pg/ml peptide. Each trace represented the average of3 replicates
and
was corrected by referencing to blank buffer injections. Responses were also
obtained when MT was injected alone and in the presence of hinge peptide
SCI~KSCC (aa 28-34 of SE(~ ID NO: 1). The response when MT is injected at a
concentration of 2,000 p.g/ml and the response when MT (2,000 pg/ml) and
peptide
CA 02554216 2006-07-19
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(250 yg/ml) are coinjected were plotted on a graph (not shotvn). Each trace
represents
the average of 3 replicates and was corrected by referencing to blank buffer
injections.
The results indicated that a peptide representing this overlap sequence,
SCKKSCC (aa 2$-34 of SEQ ID NO: 1), bound to megalin and also disrupted the
binding of native MT (see, also, Table 3). The dose-dependent binding of this
peptide
to megalin is shown in Table 3.
The ability of the peptide to affect the binding of MT to megalin is apparent
in
which the binding of MT decreased when coinjected with peptide. In contrast,
peptides containing a lysine repeat but derived from unrelated ATPase or
apolipoprotein A-I sequences had no apparent effect, producing instead
responses that
were essentially additive (Table 3). The polybasic megalin ligand, gentamicin,
bound
megalin with aa2 affinity much lower than MT and showed no interference with
MT
binding (Table 3).
SPR analysis of binding to megalin shows that, when corrected for the
molecular mass of the protein fragments (n = 2 for each analysis), the
recombinant
full-length MT clone bound 95% as well as the native protein, the a-subunit
with an
intact conservatively substituted SCICI~SCC (aa 28-34 of SEQ ID NO: 1) region,
also
bound ~94% as well as the native M.T (see Table 4). However, the (3-subunit o
which
tlae SCKKSCC (aa 28-34 of SEQ ID NO: 1) region is divided at KK has binding
reduced to 30% of the predicted value (see Table 4).
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TABLE 3: Binding to megalin by polybasic peptides and gentamicin and
interference with binding of native MT using surface plasmon resonance
techniques
Peptide or ReagentSequence Binding to Effect on
MT
Megalin Binding to
Corrcpared Megalin
With
MT
MT sequence overlapSCKKSCC (aa Competitive
2$-34 with
of pcptid~s 3 of SEQ ID NO: Affinity ~ MT binding
and 4~ 1) MT
v-ATPase (3-subunitCLQKFEKKINQSPY Amity MT Additive to
MT
peptide with EKR (SEQ ID binding
KK motif NO: 5)
Apolipoprotein-A-1ALEEYTKKLNTQ Affinity Additive to
MT MT
peptide with (SEQ ID NO: bindin
ILK motif 6)
Gentamicin Affinity < Additive to
MT MT
binding
TABLE 4: Binding to megalin by recombinant proteins derived from mouse MT and
interference with binding of the native protein using SPR techniques
Clone % Predicted Bindin b
SPR
Native full-length 100 (positive control)
MT
MT full-length recombinant 95
MT a-subunit recombinant 94
MT (3-subunit recombinant 30
EXAMPLE 4: PROTEIN-RECEPTOR BINDING IN MEMBRANE VESICLES
AND DISPLACEMENT BY ANTIBODIES STUDIED BY FLOW CYTOMETRY.
A. Prepa~~ation f Flzroropho~e-Conjugated MT.
MT was conjugated to Alexa Fluor 594, FIuorX, or Cy3 (Molecular Probes)
following the supplier's protocols. Because MT is a very small protein,
unreacted dye
was removed by dialysis against PBS at pH 7.4 in Slide-A-Lyzer dialysis
cassettes
having 3,500-lcDa molecular mass cutoff (catalog no. 66330, Pierce) rather
than with
the use of the columns provided in the manufacturer's Icit.
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B. Flow cytometry analysis of the displacenrerrt offluorescently labeled
MT frorrr brush-border rraernbrarnes by anti-receptor arrtisera, peptides arad
ligarrds.
Rat renal cortical brush-border membrane vesicles were isolated by
magnesium precipitation techniques as described previously (Batuman V, et al.,
1990
Anr ,JPI?ysiol Rer7al Fluid Elects°olyte Phyi~iol 258: F 1259-F 1265; I-
Iannnond TG et al,
1994 ,~r7a JPhysiol Renal Flz~id Electrolyte Phy.s~iol 267: FS 16-F527; Sahal
i, cited
above.) and incubated with fluorescently conjugated MT and receptor antisera..
The
binding of MT was investigated in the presence of 100-to 3,300-fold dilutions
of anti-
cubilin or anti-megalin polyclonal antibodies that recognize the holoprotein.
Antibodies to the ATt receptor and anti-NKt peptide antibodies were chosen as
negative controls for nonspecific interference by binding because they bind
brush-
border membrane vesicles at the same titer as the anti-megalin antisera.
Binding of FIuorX (Amersham Biosciences, Piscataway, NJ)-conjugated MT
was analyzed by flow cytometry using a FACStar Plus flow cytometer (Becton
Dickinson Immunocytochemistry, San Jose, CA) to collect data files of 2,000
observations/sample. All antisera were used at 1:1,000 dilutions, which
represented
peal: binding on dilution curves. Synthetic peptides were used at
concentrations of
400yg/ml, which was enough to inhibit significantly the binding of MT when
observed by SPR. For consistent comparison, the known megalin ligand j~2-
microglobulin was also used at 400pg/ml.
C. Resrcl is
The binding of fluorescent MT to vesicles was readily detected (FIG. 3). The
observed $uorescence is shown for the control (MT alone) and MT in the
presence of
anti-cubilin, anti-megalin, and anti-NK1-peptide antibodies. Data files
of2,000
observations/sample were collected. The addition of anti-megalin antibodies
was
able to displace nearly all bound MT (no antibody, MT binding 161 ~ 4
fluorescence
units, n = 5; anti-megalin antibodies 14 ~ 5, ra=5, P<0.01 by ANOVA and
Scheffe, as
well as on each individual run by Kolgomorov-Smirnov, cited previously.
Antibodies
to cubilin had a small but significant effect on binding (146 ~ 5, n = 5, P <
0.05 by
Kolgomorov-Smirnov compared with no antibody). However, antiserum to the
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unrelated NK1 receptor (a negative control) had little or no effect (150 X21,
rr = 5, P >
0.05 compared with no antibody).
The binding of fluorescent MT to freshly prepared rat brush-border membrane
vesicles was readily detected [3~1 arbitrary fluorescence units in unstained
control
vesicles, compared with 534 ~ 180 when vesicles were labeled with fluorescent
MT, rr
= 6, means ~ SD] (FIG. 4). The addition of peptide 2 (amino acids 10-25 of SEQ
ID
NO: 1), at equimolar concentrations to the fluorescent MT reduced MT binding
to 434
X156 (P < 0.05 by ANOVA and Scheffe as well as on each individual run by
Kolgomorov-Smirnov) (Young IT. 1977,IHistochern Cytochern 25: 935-941), with
further reduction to 336 ~ 97 when fluorescent MT competed with equimolar
overlap
peptide SCKKSCC (aa 28-34 of SEQ ID NO: 1; P < 0.01). The known megalin
ligand (32-microglobulin, which has been demonstrated to compete with MT in
live
rat studies (Bernard A, et al, 1988 Kidney Irrt 34: 175-185), competed with
fluorescent MT at equimolar concentrations (394 ~ 128, r~ = 6, P < 0.05),
whereas
antibodies to the unrelated AT1 receptor (a nonspecific control) had little or
no effect
(513 ~ l57). Fluorescently conjugated MT competes directly with equimolar
unlableled MT (398 ~I I4, r~ = 6, P < 0.05 compared with MT, no competition).
Solubility limitations prevented study of higher competing concentrations of
peptides.
EXAMPLE 5: CELL CULTURE STUDIES BY FLUORESCENCE MICROSCOPY
~J. Preparation of'C'ell C.'ultacr°c,s
Except as noted, experiments were conducted using immortalized yolk sac
cells from the Brown Norway rat (BN 16) (Le Panse S. et al, 1997 E~p Neplzr~7
5:
375-383). An apical brush border and a specialized endosomai pathway similar
to the
renal proximal tubule, including abundant expression of megalin and cubilin,
characterize these cells. The cells were grown in DMEM (GIBCO/Invitrogen,
Carlsbad, CA) supplemented with 10°~o fetal calf serum and 50 EtgJml
streptomycin or
ciprofloxacin. Cells were passaged every 4 days with a split ratio of 10:1.
Madin-
Darby canine kidney (MDCK) cells were grown in a modif ed minimal essential
medium as described in American Type Culture Collection (Manassas, VA)
protocols.
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B. ~fil Uptake by BN 16 Cells Analyzed By Epifh.rorescence And ~'oufoeal
Microscopy.
Uptake experiments were performed with confluent monolayers cultured in
eight-chamber glass slides (Nalge Nunc, Naperville, IL). The BN-16 cells were
cultured on chambered slides until confluent (~10-18 h). The monolayers were
washed twice with cold PBS and allowed to equilibrate at 4°C in a cold
room. The
labeled MT in DMEM containing 0.01% ovalbumin was added at concentrations
ranging from 0.075 to 12~.M. After incubation at 37°C for 20 min, the
medium was
removed and the cells were washed successively with PBS/0.1% ovalbumin (2X)
and
PBS before being fixed and mounted. The slides were examined by use of a
fluorescence microscope (Leica DMR, Basel, Switzerland) equipped with a color
video camera (Sony 3CCD). This experiment was used to select a concentration
of
1.O~M for subsequent experiments involving the labeled ligand.
In a time-dependent uptake experiment using labeled MT, cells were prepared
as before but incubated with 1.0 ~M ligand for intervals of 5, 15, 30, and 45
min. In
receptor colocalization experiments, the cells were permeabilized with Triton
X-100
(0.05% in PBS) and treated with the appropriate primary and secondary
antibodies
after fixation. The primary antibodies included anti-megalin, anti-cubilin,
anti-TfR,
and anti-giantin. To follow the internalization of MT, Alexa-labeled MT was
added
at concentrations of 1.0 or 6.O~M and the cells were incubated in the cold for
intervals
ranging from 5 to 45 min before being fixed.
Based on these experiments, confluent monolayers were washed with PBS and
allowed to equilibrate in a cold room with labeled MT (2.5 ~M) forlh at
4°C. After
being washed with PBS, the cells were treated with warm DMEM containing 2.5 pM
unlabeled MT and 0.01% ovalbumin and immediately transferred to an incubator.
Cells were fixed at intervals of 5, 15, and 45 min. Finally, the cells were
permeabilized and incubated with the appropriate primary and secondary
antibodies to
localize megalin, cubilin, and TfR.
C. MT Uptake By MDGK Cells Analyzed By Confocal Microscopy.
MDCK cells were cultured on chambered slides until confluent (~2 days). The
monolayers were washed twice with PBS and treated with labeled MT in DMEM
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containing 0.01% ovalbumin. The labeled MT was added at a concentration of 1.0
~M. A$er incubation at 37°C for 30 min, the medium was removed and the
cells were
washed successively with PBS/0.1% ovalbumin (2x) and PBS before being fixed
and
mounted. To assist in visualization of the cells, some samples were
permeabilized
with Triton X-100 (0.05% in PBS) and stained with DAPI. After a preliminary
examination with a fluorescence microscope as described above, confocal
microscopy
was carried out with a Leica TCS equipped with a DMR inveuGed microscope and a
63/1.4 objective. Image processing was performed with the use of the Leica's
online
Scanware software. Numeric images were processed with the use of Scion Image
and
Photoshop 5.0 software.
1?. MT Uptcrlre By BN 16 Cells Ar~ah~zed By Flt~~~~ Cytornetr;y.
Uptake experiments used FluorX-or Cy3-labeled MT and were performed
with confluent monolayers cultured in 96-well plates. In preliminary
experiments, MT
uptake was determined to be linear for at least 3 h and exhibited Close-
dependent
saturation. The concentration producing halF maxhnal uptake was ~5 ECM.
Inhibition
experiments were performed as Follows. The canfluent monolayers were washed
with
serum-free DMEM and allowed to equilibrate for 2h at 37°C. The cells
were then
incubated with 5 ~iM labeled MT and any inhibitor for 1-2.Sh at 37°C.
Incubations
were performed in DMEM containing 0.1% ovalbumin to reduce nonspecific
binding.
The cells were washed several times with P.BS, acid-washed to release membrane-
bound proteins, released with trypsin, and washed several more times with PBS.
In
this state they could be analyzed immediately, without fixing, by Ilow
cytometry
analysis. The positive control was labeled MT without added inhibitor; the
negative
control was unlabeled MT. Inhibitor concentrations were generally 10-I00x
greater
than the concentration of labeled >'\%IT.
E. ReszrlJs
Epifluorescence and confocal microscopy analysis of the time-dependent
uptake of fluorescently labeled MT in BN-16 cells and colocalization with
megalin
and cubilin were performed as follows. Comlluent monolayers of BN-16 cells
were
incubated with fluorescently labeled MT at 37°C. After 30 min,
fluorescence
microscopy revealed that much MT could be found in the cells in a granular
form,
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consistent with MT uptalee into endosomes. To follow cellular uptake more
closely,
BN-16 cells were incubated with labeled MT at 4°C and chased with
unlabeled MT
for variable intervals. At 4°C, MT bound to the surface but did not
enter the cells,
whereas incubation at 37°C afterward led to uptake. Colocalization with
antibodies to
the transferrin receptor, an early endosomal marleer, indicated that MT
entered the
early endosomes within 15 min but passed beyond them in <45 min.
Colocalization ofMT with both megalin and cubilin was demonstrated by
using receptor antibodies in conjunction with a fluorescent secondary
antibody. At
4°G, megalin, cubilin, and MT were colocalized on the surface, whereas
after 15 min
at 37°C they had all migrated to the early endosomes (data not shown).
After 45 min,
little evidence for colocalization remained (data not shown). No
colocalization was
observed with antibodies to giantin, an unrelated protein found in the Golgi
apparatus
and used as a negative control (data not shown).
Using confocal microscopy, results with this higher resolution method for
colocalization confirmed that MT and megalin or cubilin were colocalized.
Similar
patterns of distribution are observed for fluorescent MT and antibody (data
not
shown). As a negative control, MDCK cells were examined for evidence of MT
uptake. These cells do not express cubilin or megalin, and in fact we found
that they
did not import MT at all, demonstrating that ordinary membrane diffusion (of
free dye
or of conjugated M.T) cannot explain the results with BN-16 cells.
EXAMPLE 6: :MT UPTAKE IN CULTURED CELLS AND INHIBITION BY
ANTIBODIES, LIGANDS, AND PEPTIDES STUDIED BY FLOW CYTOMETRY.
Consistent with a receptor-mediated process, MT uptake may be saturated,
inhibited by receptor ligands and by MT model compounds, and inhibited by
receptor
antibodies. To determine the cellular uptake of fiuorescently labeled MT and
inhibition by known megalin ligands, we began with dose-and time-dependent
uptake
studies. Incubation of BN-16 cells with 0-80 pM fluorescently labeled MT for 3
h,
followed by flow cytometry analysis, demonstrates that MT uptake is saturable
and
that MT concentrations of 4-5 pM produce half maximal uptake. Under these
conditions, the uptake of labeled MT was easily distinguished against
baclcground
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signal (FIG. 6A). Uptake of MT by BN-16 cells was then demonstrated to be
linearly
time dependent at doses above and below the half maximal binding concentration
(FIG. 6B). The addition of (32-microglobulin reduced MT uptake in a dose-
dependent
manner across a broad range of concentrations of both MT and (32-microglobulin
(FIG.6C~.
Incubating BN-16 cells with anti-megalin antibodies before adding fluorescent
M'l' greatly reduced the uptake of MT in a dose-dependent manner (F1G. 7A)
(unstained cells geometric means 5 ~ 0, increases to 132 ~ 18, >7 = 5, P <
0.05, means
~ SD, with antimegalin antisera at 1:100 dilution 17 ~ 2, at 1:330 dilution 40
~ 3, at
1:1,000 dilution 90 ~ 2, r~ = 5, P < 0.05). At the same titer, anti-cubilin
antiserum had
a smaller although significant effect (at 1:100 dilution 48 ~ 7, at 1:330
dilution 96 ~ 6,
at I :1,000 dilution 116 ~ 12, n = 5, P < 0.05). AT1 antiserum, in contrast,
had no
effect (at 1.:100 dilution 161 ~ 14, at 1:330 dilution 149 ~ 9, at 1:1,000
dilution 11 '1 ~
19, n = 5, P > 0.05). Antibodies against megalin and cubilin had an additive
effect (at
1:100 dilution 7 ~ 4, at I :330 dilution 23 ~ l, at 1:1,000 dilution 62 ~ 5, n
= 5, P <
0.05 against both anti-megalin and anti-cubilin alone). The effect of
antibodies on MT
uptake was not observable unless ovalbumin was used to reduce nonspecific
effects.
The effect of the synthetic MT-derived peptides (FIG. 1) on MT uptake could
be observed when nonspecific binding was carefully excluded. The addition of
ovalbumin, which reduces the nonspecific binding of proteins to BN-16 cells
(Verroust PJ. et al, 2002 Kidney Int 62: 745-756), unmasked the differential
effects of
these peptides on the uptake of MT. The greatest effect was produced by
peptides
containing the KK sequence of the interdomain region of MT: peptides 2 and 4
and
the SCKKSCC overlap peptide reduced binding (FIG. 7B) (control 17 ~ 1
fluorescence units geometric means ~ SD, f-r = 5, MT alone 411 ~ 41, peptide 2
398 ~
84, n = 5, P > 0.05, peptide ~ 349 ~ 65, rt = 5, P < 0.05, overlap peptide 280
~ 51, r~
5, P < 0.05). When ovalbumin was not included in the incubation medium,
however,
the differences among the peptides were insignificant. Increasing the ratio of
peptide
to MT above the 1:20 ratio shown here to look for a larger effect was
prevented by
peptide solubility.
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Uptake was also inhibited by unlabeled MT and recombinant MT domains
(FIG. 7G'). As expected, unlabeled recombinant mouse MT (shown to adopt the
native structure; Atrian et al, cited above) competed strongly with the
labeled MT
(unstained BN cells 4 ~ 0 fluorescence units, geometric mean ~ SD, n = 4, MT
alone
129 ~l 8, n = 5, P < 0.05 compared with unstained, recombinant MT competition
17 ~
1, ~ = 4, P < 0.05). Interestingly, the separated recombinant MT domains did
not
produce equivalent effects: whereas the recombinant a-domain with an intact
interdomain motif inhibited approximately as well as intact M'T (9 ~ l , n =
4, P < 0.05
compared with MT alone), the ~-domain with a disrupted interdomain motif had a
much smaller eFfect (68 ~ 5, l~ = 4, P < 0.05 compared with both MT alone and
recombinant MT) (FIG. 7C.").
In summary, Examples I-6 provide three lines of evidence that megalin binds
MT and that this is the predominant mechanism of uptake of MT and its
conjugated
heavy metals in the kidney. The hinge region of MT, based around the highly
conserved lysine repeat, is one critical peptide sequence for the MT megalin
binding
interaction. MT fragments and mutants truncating or altering the hinge region
prevent
megalin-medicated renal uptake of conjugated heavy metals and secondarily
diminish
or abolish heavy metal renal tubular damage.
While the examples above provide evidence consistent with megalin being the
predominant uptake mechanism for M.T, we cannot exclude a role for other
pathways,
especially a role for cubilin. The antibody binding data on both brush-border
membrane vesicles and BN-16 cells shows an effect of anti-cubilin antiserum on
MT
binding and uptake. Megalin is a molecular chaperone for cubilin (Verroust PJ.
et al,
2002 Kidney Int 62: 745-756, Verroust PJ and Isozyraki R. 2001 C,'zrr°r
O~aisa Neplzs°ol
Ilypertens 10: 33-38), so the colocalization studies, not surprisingly,
demonstrate
colocalization of NIT with both eubilin and megalin during the early steps of
internalization and uptake. The only data collected against a role for cubilin
in MT
binding and uptake are the direct studies of molecular interactions using SPR
techniques. Although other known ligands of cubilin bound in control studies,
paulial
denaturatian of cubilin, which is inevitable during its purification, may mask
binding.
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Thus, cubilin may also play a role in MT uptake in the proximal tubule of the
kidney
and other cubilin/megalin-expressing epithelia such as in the placenta.
EXAMPLE 7: NOVEL MUTANT MT-HEAVY METAL COMPLEXES
A. Ps~epaf°ation of f~econabinant mouse metallothionein I, and a
and /3
subunits.
Recombinant fragments of mouse MT were produced successfully and
reproducibly according to the procedures of Atrian et al, cited above. This
approach
seems more practical than site directed mutagenesis, as most attempts to
produce
recombinant MT have been characterized by very low yields or by mixtures of
several
short cleavage fragments of the MT molecule (Sabolic I, et al, 2002. Am
J~'hy.siol
Renal Phvsiol 283: P1389-F1402; Suzuki-Kurasaki M, et al, 1997 JHistochc~n~
Cytoelaem 45: 1493-1501). This problem was resolved by making recombinant MT
subunits in E. coli using a GST fusion vector followed by thrombin cleavage to
release the free MT subunit (see, Atrian et al, cited above). The thrombin
cleavage
leaves three amino acids, specifically SCM derived from the C-terminus of the
GST,
on the N-terminus of the product.
It is theorized that the critical binding site on MT is the intradomain SCK-
KSCC region (aa 28-34 of SEQ ID NO: 1) with SCK representing the C-terminus
end
of the (3-subunit, and KSCC the N-terminus start of the a subunit. The
recombinant a
subunit therefore has a conservative GST derived SCM substitution for SCK on
its N-
terminus, leaving the postulated critical SCKKSCC sequence (aa 28-34 of SEQ ID
NO: 1 ) essentially intact. The recombinant ~i-subunit starts with SCM- and
ends in
SCK rendering the postulated critical SCKKSCC (aa 28-34 of SEQ ID NO: 1 )
disrupted. The full-length recombinant MT has an intact SCKKSCC (aa 28-34 of
SEQ ID NO: 1 ) sequence as well as an addition N-terminal SCM-. Inductively
coupled plasma (ICP) analysis of the zinc and platinum content of the
recombinant
subunits proved them to be at the predicted heavy metal content to within the
error of
the methods. Protein concentrations were assayed by the Bradford method
(Pierce
Biotechnology, Roclcford, IL).
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B. Alkalitze phosphatase conjugated metallothionein and subutZits.
Metallothionein, recombinant a subunits and recombinant (3 subunits
conjugated to alkaline phosphatase as a reporter of uptake. To conjugate gut
isoform
alkaline phosphatase one mg of each protein was dialyzed overnight into 0.1 M
sodium carbonate/bicarbonate buffer at pH 9.8, reacted with activated alkaline
phosphatase, according to the method of the AP Labeling lcit from Roche
Diagnostics
(Mannheim Germany). The conjugated proteins were dialyzed overnight into
phosphate buffered saline prior to use. Because MT is a very small protein,
unreacted
dye was removed by dialysis against PBS at pH 7.4 in Slide-A-Lyzer dialysis
cassettes having 3500 IcD molecular weight cut off (Pierce, cat. no. 66330,
Roclcford
IL), rather than using the columns recommended in the manufacturer's kit.
C. Cot jatgation of casplatin into metallothioneitz and subunits.
To replace all heavy metals in metallothionein with cisplatin, commercial
metallothionein I (Sigma Chemical, St Louis MO) was combined in a ratio of 5:3
1 S metallothionein to cisplatin by weight in l OmM HEPES buffer, and
incubated at 37°C
for 48 hours. The product was dialysed against PBS or carbonate buffer as
appropriate for toxicity and uptake experiments.
To remove cadmium in commercial metallothionein and replace with zinc,
Smg of mouse metallothionein I was put into solution in l OmM HEPES buffered
to
pH 3.0 degassed with dry nitrogen to strip all heavy metals. Then zinc was
added by
dialysis against a buffer containing 2mM beta-mercaptoethanol, and 1mM zinc
chloride in l OmM HEPES pH 7.4. All metallothionein reagents had their
platinum
contents confirmed by inductively coupled plasma.
EXAMPLE 8: METALLOTHIONEIN-CISPLATIN COMPLEXES, THEIR
NEPHROTOXTCITY AND TOXICITY RELATIVE TO CISPLATIN ALONE.
CD-1 mice received a single intraperitoneal injection of 20mg/kg cisplatin
either alone or conjugated to full length or recombinant metallothionein
subunits
prepared as in Example 7. The mice were sacrificed 48 hours later and blood
collected
for creatinine analysis as an index of renal function. Aliquots of kidney and
liver
tissues and a set aside aliquot of the blood were harvested for subsequent
analysis of
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platinum content by inductively coupled plasma. The serum creatinine in mg/dl
was
assayed using a plate based colorimetric assay (Cayman Chemical, Ann Arbor MI)
on
blood drawn 48 hours after cisplatin treatment of mice.
The results are demonstrated in FIG. 7. The creatinine values were
0.055+0.022 in the group of mice treated with phosphate buffered saline (PBS)
vehicle (mean + standard error, n=5); following cisplatin treatment (cis)
creatinine
increased significantly to 0.105 + 0.056 mg/dl; metallothionein alone (ZnMT)
resulted
in creatinine values of 0.068 + 0.028; metallothionein carrying cisplatin (MT-
cis)
0.083 + 0.053; alpha subunit carrying cisplatin (alpha cis) 0.069 + 0.030; and
beta
subunit carrying cisplatin (beta cis) 0.060 + 0.023. The groups significantly
different
from phosphate buffered control at p<0.05 level by one-way ANOVA and Tulcey's
post-hoc comparison are the cisplatin alone and MT-cis groups. The beta
cisplatin
group is significantly less than the cisplatin alone group, demonstrating
renal
protection with this reagent.
EXAMPLE 8: CYTOTOXICITY AND ANTI-TUMOR EFFICACY IN VITRO OF
METALLOTHIONEIN-CISPLATIN COMPLEXES.
The cytotoxicity and anti-tumor efficacy of the MT-Cis complexes of Example
7 were determined by testing for uptake of metallothionein and its fragments
into lung
cancer (small cell carcinoma), invasive transitional cell bladder cancer (J82)
cells and
ovarian carcinoma cells (OVCAR-3), and conducting an assay of induction of
apoptosis as evidenced by caspase 3 levels.
A. Culture of cancer cell lines.
J82 cells, a bladder transitional cell carcinoma cell line, was cultured in
EMEM alpha medium with 10% fetal calf serum and cisporofloxacin. DMS53, small
cell lung carcinoma of the lung was cultured in Waymouth's MB 752/1 culture
media
with 10% fetal calf serum and ciprofloxacin. Cells were grown to near
confluence in
T75 flasks and then split into 96 well plates for study. OVCAR'3 cells were
cultured
in DMEM with 10% fetal calf serum and pen/strep.
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B. Statistics.
Data are expressed as mean + standard error of the mean throughout these
examples. Statistical analysis was performed by analysis of variance and
Tukey's
post hoc comparison.
C. Metallothio~ei~r uptake by cells a~aalyzed by abso~~ptioh spectoomet~y
in lung cafzce~° cells (small cell carcinoma).
Uptake experiments using alkaline phosphatase conjugated to metallothionein
and its recombinant subunits were performed with confluent monolayers cultured
in
96-well plates. In preliminary experiments, metallothionein uptake was linear
for at
least 4 hours and exhibited dose-dependent saturation. Experiments are
performed as
follows. The confluent monolayers are washed with serum-free media and allowed
to
equilibrate at for two hours at 37 °C. The cells are then incubated
with 10 pM labeled
metallothionein for 4.0 hours at 37 °C. The cells are washed several
times with PBS,
acid-washed to release membrane-bound proteins, and washed several more times
with PBS.
Alkaline phosphatase activity is assayed in the wells by addition of alkaline
phosphatase yellow (pNPP) liquid substrate system (Sigma Chemical Company St
Louis, MO.) and incubation. Alkaline phosphatase is then analyzed immediately
on a
plate reader using absorbance as the endpoint.
As illustrated by FIG. 8, the absorbance of the alkaline phosphatase
conjugated to the metallothionein and its subunits following uptake into lung
small
cell carcinoma cells for 4 hours was 0.031+0.005 in phosphate buffered saline
vehicle
(mean + standard error, n=6). Following O.SmM cisplatin treatment for 4 hours,
this
increased significantly to 0.068 + 0.015. Metallothionein alone was 0.25 +
0.04; and
metallothionein carrying O.SmM cisplatin was 0.46 + 0.03. The MT-a subunit
carrying was O.SmM cisplatin 0.31 + 0.06; and MT-(3 subunit carrying 0.5 mM
cisplatin was 0.37 + 0.06.
All other groups are significantly different from phosphate buffered saline
control at p<0.05 level by one-way ANOVA and Tukey's post-hoc comparison in
all
groups compared to phosphate buffered saline other than the cisplatin group.
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D. Caspase-3 activation ire cell treated with cisplatirr conjugated to
nretallotlZiorzein arid its sZrbunits in lung cancer cells.
To determine metallothionein and its subunits induce caspase-3 activation,
commonly associated with induction of cell death by apoptosis, cells were
treated as
in the uptake protocol above and the caspase-3 therein was assayed.
CaspACETM FITC-VAD-FMK in situ marker (Promega, Madison WI) is a
fluorescent analog of the pan caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-
alanyl-aspartyl-[O-methyl]-fluoromethyllcetone). The fluoroscein
isothiocyanate
(FITC) group has been substituted for the earbobenzoxy (Z) N-terminal blocking
group to create the fluorescent apoptosis marker. This structure allows
delivery of the
inhibitor into the cell where it irreversibly binds to activated caspases. The
FITC label
allows for a single-reagent addition to assay activated caspase-3 activity irr
situ.
As shown in FIG. 9, the absorbance of the activated caspase-3 specific probe
was 0.007849+0.00483 in phosphate buffered saline vehicle (mean + standard
error,
n=6). Following O.SmM cisplatin treatment for 4 hours, this result increased
significantly to 0.012656 + 0.008571, p<0.05. Metallothionein alone was
0.036802 +
0.015034; metallothionein carrying O.SmM cisplatin was 0.015741 + 0.011293; MT-
a
subunit carrying O.SmM cisplatin was 0.027545 + 0.023401; and MT-/3 subunit
carrying 0.5 mM cisplatin was 0.019419 + 0.011565. All other groups were
significantly different from phosphate buffered saline control at p<0.05 level
by one-
way ANOVA and Tukey s post-hoc comparison.
These results demonstrate that cisplatin is equally or more effective in
inducing apoptosis in small cell carcinoma cells when conjugated to
metallothionein
fragments compared to treatment with unconjugated cisplatin.
E. Metallotlaiorzeirr arptake by cells analyzed by absorption spectrometry
in bladder cancer cells (.I~2 trarfrsitio>zal cell carcirronra)
Uptake experiments using alkaline phosphatase conjugated to metallothionein
and its recombinant subunits, were performed with confluent monolayers
cultured in
96-well plates as for the other cell lines. As shown in FIG. 10, the
absorbance of the
alkaline phosphatase conjugated to the metallothionein and its subunits
following
uptake into lung small cell carcinoma cells for 4 hours was 0.051+0.006 in
phosphate
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buffered saline vehicle (mean + standard error, n=6). Following O.SmM
cisplatin
treatment for 4 hours, this changed to 0.082 + 0.016; metallothionein alone
0.27 +
0.04; metallothionein carrying O.SmM cisplatin 0.48 + 0.03; alpha subunit
carrying
O.SmM cisplatin 0.33 + 0.04; and beta subunit carrying 0.5 mM cisplatin 0.39 +
0.06.
All other groups are significantly different from phosphate buffered saline
control at p<0.05 level by one-way ANOVA and Tukey's post-hoc comparison in
all
groups compared to phosphate buffered saline other than the cisplatin group.
F. Caspase-3 activation ifz cell treated with cisplatin conjugated to
metallothiohein acrd its suburzits ih bladder cancer cells.
To determine whether metallothionein and its subunits induce caspase-3
activation, commonly associated with induction of cell death by apoptosis,
cells
treated as in the uptake protocol above had caspase-3 assayed. As illustrated
in FIG.
11, the absorbance of the activated caspase-3 specific probe was 0.017~
0.007in
phosphate buffered saline vehicle (mean ~ standard error, n=6). Following
O.SmM
cisplatin treatment for 4 hours, this increased significantly to 0.038~ 0.016,
p<0.05;
metallothionein alone 0.019+ 0.008; metallothionein carrying O.SmM cisplatin
0.042~
0.017; alpha subunit carrying O.SmM cisp(atin 0.053-0.021; and beta subunit
carrying
0.5 mM cisplatin 0.043+0.018.
All other groups are significantly different from phosphate buffered saline
control at p<0.05 level by one-way ANOVA and Tukey's post-hoc comparison
except
metallothionein alone.
In a second performance of the bladder cell studies, J82 transitional cell
carcinoma cells were grown in 96 well plates. Following growth to near
confluence
the cells were changed to serum free medium, and 0.5 mM cisplatin in serum
free
medium added either alone or conjugated to the beta subunit of
metallothionein. The
beta subunit of metallothionein was prepared recombinantly, and cisplatin was
conjugated by incubation at 37°C for 48 hours. Residual excess
cisplatin was removed
by overnight dialysis against phosphate buffered saline. After 4, 8 or 22
hours of
incubation in cisplatin, the cells were washed with phosphate buffered saline
and
activated caspase -3 labeled by addition of a fluorescent cell permeable
peptide which
binds specifically to caspase-3. After 30 minutes labeling with the caspase-3
peptide,
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cells were again washed with phosphate buffered saline, trypsinized off the
plates and
caspase-3 activation assayed by flow cytometry using fluorescence of the
fluorescent
peptide as a reporter. The results in FIG. 14 show that cisplatin administered
conj ugated to the beta subunit of metallothionein induces at least as much
activation
of caspase-3, and hence cell death by apoptosis as cisplatin alone.
G. Ovarian Carcitzorna (OhCAR-3) cells take up MT
To determine if OVCAR3 cells take up MT, the cells were incubated in
fluorescently labeled MT. OVCAR3 cells were grown to confluence in 96 well
plates,
and serum starved before exposure to 40p,M Cy3-fluorescently conjugated MT for
1,2
and 4 hours. Cells were washed, surface binding released with acid, and
trypsinized
for flow cytometry analysis of MT uptake.
Compared to unstained cells (0~0) fluorescent units, the MT was taken up
significantly by 1 hour (10+3fluorescent units, n=4, p<0.05 compared to
unstained), 2
hours (13+3fluorescent units, n=4, p<0.05 compared to unstained), or 4 hours 1
hour
(16+2 fluorescent units, n=4, p<0.05 compared to unstained) as illustrated in
FIG. 12.
H. Cisplatin aid MT eisplati~ kill OVCAR cells.
To determine if our OVGAR3 cells are a good model for cisplatin efficacy, the
cells were exposed to cisplatin. As in the protocols above the confluence
cells in 96
well plates were serum starved for 2 hours, prior to addition of cisplatin, MT
complexed cisplatin, or the MT carrier alone. Cell death was assayed by
trypsinizing
the cells off the plates, staining with propridium iodide and assaying loss of
cell
membrane integrity by flow cytometry.
Preliminary studies on dose and time course showed 250pM for 24 hours
results in significant cell death, and these parameters were adopted for these
comparison studies. As expected, cisplatin kills OVCAR3 cells (untreated
OVCAR3
cells 20+5 fluorescence units, geometric mean ~ standard deviation n=4,
cisplatin
alone 53+2, n=4, p<0.05 compared to untreated, cisplatin complexed to MT 41~5,
n=4, p<0.05, and MT alone 20+6, not significant). The results are demonstrated
in
FIG. 13 and demonstrate that MT is taken up into OVCAR3 cells and that MT
complexed cisplatin has almost the same efficacy in cell killing as free
cisplatin.
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In summary, the above Examples 7 and 8 demonstrate that complexes of the
beta subunit of metallothionein and cisplatin are significantly less
nephrotoxic that
cisplatin alone. Metallothionein-cisplatin complexes retain cytotoxic anti-
tumor
efficacy in vitro.
All documents, including I~lassen et al, Af~a. J. Phsiol. Reiaal. Physiol,
2~7:F393-403 (May 4, 2004), the priority document, and public databases cited
within
this specification are incorporated herein by reference.
62
DEMANDES OU BREVETS VOLUMINEUX
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COMPRI~:ND PLUS D'UN TOME.
CECI EST L,E TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
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THAN ONE VOLUME.
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