Note: Descriptions are shown in the official language in which they were submitted.
WO9l/17761 PCT/US9l/03291
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2 0 8 2 ~ o r¦
TARGETED PROTECTION FROM CYTOTOXINS
Ba~kground of the Invention
Chemotherapeutic agents currently available for
tr~atment of tumors can be unsuccessful because they
05 lack tumor specificity. The use of galactosamine has
been e~plored in the treatment of primary liver cancer
(hepatocellular carcinoma) because it is a highly
selective liver to~in ia vitro and n vivo. The
selectivity is due to elevated intrahepatic levels of-- ------ -
two enzymes of the galactose metabolic pathway,galactokinase, and UDP-glucose:galactose-l-P-
uridyltransferase (Bertoli, D. and Segal, S. (1966)
Biol. 5hem. 241:4023 and Cuatrecasas, P. and Segal, S.
(1965) J. BiQl~ Çher~ ~40:2382), that allow :
galactosamine to be metabolized as a galactose analog
(Keppler, D. and Decker K., (1969) Eu~. J. ~iochem~ ~ :
10:219). This eventually leiads to trapping and
depletion of intracellular uridine intermediates in
hepatocytes a~d hepatocyte-derived cells (Keppler,
; 20 D.O.R., et al. ~1970) E~ _ ~L - L~ k m_ 17:246)- :
However, high doses of galactosamine sufficient to
destroy hepatoma cells results in toxicity to normal
hepatocytes. ~t has been shown that a galactosamine
antagonist can be targe~ed to hepatocytes,
25 specif ically protecting them f rom galactosamine
to~icity ;La ~i~Q (Wu, G.Y., ~; ~,. (1988) J. l~iol
4719).
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WO91/177~ PCT/US91/03291
A method of protecting normal cells n v o from
the cytotosic activity of the chemotherapeutic agents
may alle~iate the problems of to~icity and enhance the
effectiveness of these agents.
O S Summa ry o f ~hQ~D~
This invention pertains to a method of selective-
ly protecting normal cells from the cytotosic effects
of a chemotherapeutic cytoto~in directed ayainst
diseased cells such as tumor cells. According to the
1~ method, the chemotherapeutic cytoto~in is administered
in con~unction with, or subsequent to,--administration
of a~ antagonist-conjugate. The antagonist-conjugate
comprises an antagonist of the cytoto~in coupled to a
cell-speci~ic binding agent which binds to a cellular
surface component present on normal, but not on
diseased cells. The cellular surface component is
typically a receptor which mediates internalization of
bound ligands by endocytosis, such as the asialo-
~lycoprotein receptor of hepatocytes. The
cell-specific binding agent can be a natural or
synthetic ligand (for e2ample, a protein, polypeptide,
~lycoprotein, etc.) or it can be an antibody, or an
analogue th~reof,~which specificall~ binds a cel~ular
surface structure which then mediates internalization
of the bound comples. The antagonist can be complexed
with the cell-speci~ic binding agent via an
antagonist-binding agent, such as a polycation.
The antagonist-conjugate is administered ia vivo
where it is selectively taken up by normal cells via
the surface-structure-mediated endocytotic pathway.
The conjugate is administered in an amount sufficient
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WO91/~7761 PCT/U~1/03291
~3~ 2 0 ~ 7
to protect normal cells from the cytoto~ic effects of
the cytoto~in. Diseased cells which lack the cellular
surface component do not take up significant amounts
of the ant~gonist-con~ugate and are unprotected from
05 the cytotoxin. The method provides for a more
effective use of higher doses of cytoto~ins against
tumor and against other diseases by alleviating or
eliminating the to~icity to normal cells usually
associated with such therapy.
Brief De~iption o~ the~Fig~Qs
Figure l shows the or~an distribution of
- radiolabeled galactosamine antagonist-conjugate.
Figure 2 shows the effect of galactosamine
antagonist-conjugate pretreatmant on galactosamine
to~i~it~.
Detailed Desc~iption of the Inventi~
This invèntion pertains to a method of
selectively targeting an antagonist of a cytotoxin to
normal mammalian cells to protect against the adverse
effects of a therapeutic cytotosin. An antagonist-
, conjugate targetable to normal mammalian cells is used
to selectively deliver a~ antagonist to the cells n
v yo. The antagonist-conjugate comprises 2n
antagonist of the cytoto~in complexed with a cell-
specific binding agent which binds a cellular surface
component present on normal, but not diseased cells.
The antagonist-conjugate is selectively taken up by
; the normal mam~alian cells and the antagonist is
released into the cell in functional form to provide
protFction against the effects of the cy~oto~in.
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WO91/17761 PCT/US91/03291
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The cell-specific binding agent specifically
binds a cellular surface component which mediates
internalization by, for e~ample, the process of
endocytosis. The surface component can be a protein,
05 polypeptide, carbohydrate, lipid or combination
thereof. It is typically a surface receptor which
mediates endocytosis of a ligand. Thus, the surface
component can be a natural or synthetic ligand which
binds the receptorO The ligand can be a protein,
polypeptide, glycoprotein or g-ycopeptide which has
functional groups that are e~posed suf~iciently to be
recognized by the cell surface structure. It can also
be a component of a biological organism such as a
virus, cells (~.g., mammalian, bacterial~ protozoan)
or arti~icial ~arriers such as liposomes.
The cell-sp~cific binding agen~ can also be an
antibody, or an analogue of an antibody such as a
sinqle chain antibody which binds the cellular surface
component.
Ligands useful in forming the antagonist-
conjugate will vary according to the particular cell
to be targeted. For targeting hepatocytes, glyco-
proteins having exposed terminal carbohydrate groups
such as asialoglycoprotein (galactose-terminal) can be
used, although other ligands such as polypeptide
hormone~ may also be employed. E~ample~ o~
asialoglycoprot~ins include asialoorosomucoid,
; asialofetuin and desialylated vesicular stomatitis
virus. Such ligands can be formed by chemical or
enzymatic desialylation o~ glycoproteins that possess
terminal sialic acid and penultimate galactose
residues. Alternatively, asialoglycoprotein ligands
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W~91/17761 PCT/~S91/03291
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can be formed by couplin~ galactose terminal carbo-
hydrates such as lactose or arabinogalactan to
non-galactose bearing proteins by reductive amination.
For targetin~ the antagonist-conjugate to other
05 cellular surface components, other types of ligands
can be used, such as mannose for macrophages,
mannose-6-phosphate glycoproteins for fibroblasts,
intrinsic factor-vitamin B12 for enterocytes and
insulin for fat cells. Alternatively, the
cell-specifc binding agent can be a receptor or
receptor like molecule, such as an antibody which
binds a ligand (e.g., antigen) on the cell surface.
Such antibodies can be produced by standard procedures.
The antagonist-conjugate can be made by binding
15 the antagonist directly to the ligand or by binding it ~
with the ligand through an antagonist-binding agent. ~;
The antagonist-bindin~ agent comple2es the antagonist
to be delivered. Comple~ation with the antagonist
m~st be su~ficiently stable n vivo to prevent
signifisant uncoupling of the antagonist extracellu-
larly prior to internalization by the cell. However,
, the comples is cleavable under appropriate conditions
; within the cell so that the antagonist is released in
~unctional form. For e~ample, the comples can be
labile in the acidic and enzyme xich environment of
lyso~omes. A noncovale~t bond based on electrostatic
attraction between the an~agonist-binding agent and
~he antagonist provides ex~racellular stability and is
releasable under intracellular conditions.
Pre~erred antagonist-binding agents are
polycations which provide multiple bindin~ sites for
antagonists. Esamples o~ polycations include
polylysine, polyornithine or histones.
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WO91/1776~ PCT/~IS9l/03291
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The antagonist-binding component can be
covalently bonded to the ligand. A preferred linkage
is a peptide bond. This can be formed with a water
soluble carbodiimiae as described by Jung, G., et al.
05 (1981) Bi~çhem. 8iophys. Res~ CQmmun, ~Ql:599-606. An
alternative linkage is a disulfide bond.
The linkage reaction can be optimized for the
particular antagonist-binding agent and ligand used to
form the conjugate. Reaction conditions can be
design~d to ma~imize linkage formation but to minimize
the formation of aggregates of the conjugate
components. The optimal r~tio of anta~onist-bindin~
agent to ligand can be determined empirically.
Uncoupled components and aggregates can be
separated from the conjugate by molecular sieve
chromatography.
The conjugate can contain more than one
antagonist molecule or one or more different
anta~onist molecules. Preferably, ~rcm about 10-15
antagonist molecules per conjugate. The number may
vary, depending upon ~actors such as the effect on
solubility or capillary permeability of the conjugate.
The cytotosin and antagonist can be selected from
any of those effective in treatment of the disease.
~; 25 For tumor therapy, various antitumor agents ~or whi~h
an~agonists are available can be used. E~amples of
anti~umor cy~otosins and corresponding antagonists
include methotrexate/folinic acid, acetaminophen/
N~acetyl cysteine, 1,3-bis(2-chloroethyl)-l-
nitrosourea (~CNU)/N-acetyl cysteine, glutathione or
WR2721 and galactosamine~uridine monophosphate or
orotic aci~. In addltion, combinations o~ two
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WO91/17761 PCT/US91/03291
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different cytotoxins and respectiYe antagonists (which
may be the same or di~ferent~ can be used to reduce
selection o~ resistant cells.
In a preferred embodiment, the cytoto~in is
05 specific for the diseased organ or tissue. This helps
minimize to~icity of uninvolved organs~ For example,
as described, galactosamine is a highly selective .
hepatoto~in and there~ore, is preferred for treatment
of primary liver cancer such as hepatocellular
carcinoma.
In preferred embodiments, the antagonist- :
conjugate is soluble in physiological fluids. The
antago~ist-conjugate is generally administered
parenterally in a physiologically acceptable vehicle
; lS in an amount sufficient to protect normal cells
against the to~ic effects of a cytotoxin.
The invention is illustrated further by.the
: following e~emplification.
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~empli~ig~5
'20 Prep~ation of ~n~ L~ 5 ~1b~qQ~
The asialoglycoprotein, asialo~etuin (AsF) was
prepared by desialylation of bovine fetuin (GIBCO,
Grand Island, New York), using neuraminidase (Sigma
Chemica} Co., St. Louis, Missouri) to e~pose terminal
galactose residues by a modification o~ the method of
~: . Oka and Weigel ~Oka, J.A. and Wei~el P.H. (1983) ~.
~ 258:10253). Analysis o~ residual protein-
bound sialic acid by the method of Warren (Warren, ~.
~l959) J..aLo~ hem. 234:l97l) d~termined khe etuin
to be 94% desialylated.
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WO91/17761 PCT/US91/03291
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~^hQ l ~
In order to create a targetable carrier protein
with a large capacity to bind antagonist, AsF, 55 ~M
was coupled to poly-L-lysine (PL) (Sigma Chemical Co.,
St. Louis, Missouri) 470 ~M, Mr = 3600 using
05 l-ethyl-3-(3-dimethylamino~propyl carbodiimide (Pierce
Chemical Co., Rockville, IL) as described previously
(Wu, G.Y., et al. (1988) J. Biol. Chem. 263:4719).
5'-Uridine monophosphate ~UMP) (Sigma Chemical Co.,
~t. ~ouis, Missouri) was then coupled to the carrier
protein according to the method of Halloran and Parker
(Halloran, M.J. and Parker, C.W. (1966) J. Immunol.
96:373) purified by column chromatography (Wu-, G.Y.,
~t al. ~l988) J. Biol. Chem. ~~:47l9)- The conjugate
was stable at 4C for at least two weeks.
Oraan Distr bution of ~niec~ed AsF-PL-UMP Coniu~e
To determine whether the conjugate xetained its
ability to be recognized by asialoglycoprotein
receptors n vivQ both AsF and the AsF-PL-UMP
conjugate were radiolabeled with Na 125I (Amersham
Corporation, Chicago, Illinois) using a solid-phase
lactoperoxidase method as described by the
manufacturer ~BioRad). Female Sprague-Dawley rats
~220-270g) (Zivic-Miller Laboratories, Allison Park,
Pennsylva~ia) were injected intravenously with sterile
saline containing l ~g l25I-AsF, or l yg l25I
AsF-PL-UMP (based on AsF content). To determine
whether liver uptaks of the conjugate was via
asialoglycop~otein receptors, a control rat was given
1 ~9 l25I-AsF-PL-UMP plus an e~cess, l0 mg, o
unlabeled asialoorosomucoid (AsOR) to compete for
hepatic asialoglycoprotein receptors. To evaluate the
e~tent o~ non-æpecific hepatic uptake o~ coniugate, 15
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WO91/17761 PCT/US91/03~91
-9 2082~07
mg of degtran sulphate ~Pharmacia, Upssala, Sweden,
Na), an inhibitor of nonparenchymal "scavenger" 1.
receptor activity, was administered intravenously, 15
minutes prior to the conjugate injection according to
05 the method of Van der Sluijs, ~_3~l. (Van Der Sluijs~
P., Q~ l986) HepatQlogy 6:723). Another control
received both dextran sulphate and e2cess AsOR. Ten
minutes after injection of labeled protein, blood was
drawn f~om the retro-orbital ple~us and ~he animals
sacrificed. The distribution of radioactivi~y among
organs was determined by gamma counting and éxpressed
: as percent of total counts.
As shown in Figure l, for rats receiving either
conjugate or AsF, appro~imately 80% of the counts were
taken up by ~he liver. The addition of egcess AsOR
successfu~ly competed with the labeled conjugate for
hepatic asialoglycoprotein receptors resulting in
removal by liver of only 16% of the injected counts.
The inhibition of uptake of l25I-AsF-PL-UMP by
competition wi~h the excess AsOR indicates that the
: targeting of the antagonist was directed by the
asialoglycoprotein component of the conjugate. The
lack of effect of UMP injected ælone in identical
amounts and under identical conditions as for the
conjugate argues against intravascular cleavage of UMP
from the conjugate as a mechanism of the observed
protection by the conjugate.
Injection of destran sulphate, which inhibits
non-specific uptake via nonparenchymal "scavenger"
receptors, had no effect on liver uptake of the
conjugatè which.still accounted for 80% of the
injPcted radioactivity. Administration of both
de~tran ~ulphate and e~cess ~sO~ had no furtheF effect
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W~91tl7761 ~ PCT/US91/032g]
on liver uptake of the conjugate beyond that of e~cess
AsOR alone. These data indicate that neither the PL,
VMP nor the process of coniugation had altered
recognition of the AsF by hepatic asialoglycoprotein
05 receptors in the intact rat.
Potential to~icity of the conjugate itself w~s
evaluated by injecting conjugate alone at the dose and
volume used in the previou~ esperiment (34 m~/kg).
The animal was observed at various time intervals,
then sacrificed at 42 h ~the time of peak
galactosamine to2icity, and therefore of ma~imal
conjugate protection in the previous experiment).
Various oryans and tissues were removed and prepared
for histological e~amination.
The behavior of a rat receiving coniu~ate alone
showed no obvious indications of cardiopulmonary
distress or neurological deficits. No abnormalities
were revealed by histological e~amination of liver,
kidney, spleen, heart and surrounding large vessels,
lungs and trachea, brain, peripheral nerves and
ganglia, adrenal gland, lymph nodes, skeletal muscle
and adipose tissue.
f~ç~t Q~ F-PL-UMP ~oni~ate on Galaç~osamine
TQxicity
25 ~ As shown in Figure 2, the effect of the targeted
antagonis~ on galactosamine toxicity to hepatocytes
V ,VQ was determined. To allow sufficient time for
internalization of the conjugate and release of the
antagonist, the targetable antagonist-conjugate was
injected i.v. ~in 5 ml sterile saline) in female rats
as a 2 h pretreatment prior to the galactosamine
injection. Subsequently, rats were injected
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W091/17761 PCT/US91/032~1
2~2~7
intraperitoneally with 80V mg/kg galactosamine (Sigma
Chemical Co., St. Louis, Missouri), in 2.5 ml sterile
saline, pH 7.4. The mi~imum amount of conjugate
required to protect hepatocytes was determined by i.v.
05 injection of varying doses of conjugate. Using the
conjugate dose thus determined optimal ~34 mg/kg), the
ability of this antagonist conjugate to prevent
galactosamine to~icity was evaluated relative to
controls receiving i.v. injected pretreatments of
equal volumes o~ sterile saline, or saline containing
AsF or UMP in molar amounts e~uivalent to that
provided by the conjugate. Blood was withdrawn from
the retro-orbital ple~us at 24, 42, 48 and 72 h after
galactosamine injection. Hepatotoxicity was evaluated
by measurement of serum alanine aminotransferase (ALT)
l2vels (Sigma assay kit) according to the
manuacturer. All assays were per~ormed in duplicate
and e~pressed as international units per liter
(IU/l). A~dition of conjugate ~o ALT standards as
well as serum samples demonstrated that the conjugate
had no effect on the ALT assays.
A Krusk'al-Wallis test was used to evaluate
differences among the four groups (6-7 rats per
trea~ment; pretreatment ALT values averaged 38 with
S.D. of 9). Qnce a significant difference among
treatment was determine~, pair-wise comparisons were
evaluated with Wilco20n-Mann-Whitney tests ~Zar J.H.,
1984, Biostatistical analysis. Prentice-Hall,
~ngl _ ood C1iff~, ~.J.).
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W091/17761 PCT/US91/03291
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Selective uptake by the liver of conjugate in
trace amounts, demonstrated above, was also found for
this higher dose of conjugate. Because ALT valuPs
were compared at this peak 42 h (always decreasing by
05 72 h). Serum ALT values were compared at this peak
42 h time point. A Kruskal-Wallis test determined
that there were signifioant differences among the four
groups, with an alpha level of 0.01. Pair-wise
comparisons (Wilco20n-Mann-Whitney tests) determined
that animals pretreated with AsF-Ph-UMP conju~ate
e~perienced significantly less hepatoto~icity than
saline controls as measured by serum ALT levels
(pc.005). Animals that received conju~ate likewise
had significantly lower ALT values than those
re~eiving either AsF alone or UMP alone (p<.05 and
p<.~02 respectively). There were no significant
dif~erences among the three controls.
The results indicate that the AsF-P~ UMP
conjugate can be targeted to hepatocytes resulting in
protection of these cells from ga}actosamine toxicity
vivo. The lack of effect of administration of UMP
alone can be explained by the fact that uridine in the
form of the conjugate was targeted only to hapatocytes
wh;le free UMP could be dispersed by the circulation
~5 for uptake throughout the body~ Unlike ~he UMP in the
form of the conjugate, free UMP provided to ~he livar
from the circulation was evidently inadequate to
prevent galactosamine to~icity.
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AsOR-PL-UMP Coniuaate
An AsOR-PL-UMP conjugate was produced by the
method as described above. Using this conjugate, the
effect on galactosamine toxicity was determined.
05 Improvement in protection as compared to the
AsF-PL-UMP conjugate was achieved by i.v. infusion of
the AsOR-PL-UMP eonjugate o~er a 4 h period tat the
saturation rate o hepatic asialoglycoprotein
receptors). At a galactosamine dose of 500 mg~kg,
median peak alanine aminotranserase (ALT) level~ for
AsOR-Ph-UMP inused rats were 1725, compared to 3059
for saline infused controls.
AsOR-~aurin~_~on;uq~te
To achieve better protection of normal liver from
high doses of galactosamine, di~ferent antidote
conjugates were developed. The final irreversible
step in galactosamine to~icity is an influ~ of Ca+~
into the damaged cells. Since taurine can interere
with to~icity by causing intracellular sequestration
of calcium, an AsOR-taurine conjugate was developed as
described above. ~dministration of the AsOR-taurine
conjugate after ~alactosamine provided no protection.
When the conjugate was infused over a 4 h period prior
to administr~tion of 500 mg/k~ galactosamine, it
provided protection (median peak ~LT 820 compared to
2282 in saline-infused controls). Protection of
conjuga~e-pretreated animals was increased by
administering uridine (1.2 g/kg) 5 h after
galactosamine (a point when galactoæamine damage in
saline controls should be irreversible). With this
strategy, conjugate (~ uridine) treated animals
èsperien~ed median peak ALT levels of 258, compared to
729 for saline (+ uridine) treated controls.
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WO91/~776~ ~ PCT/US91/03291
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A~O~-PL-Orotic Acid Conjuqate
Following the procedure as described for the
AsF-PL-UMP conjugate, another targetable antidote
conjugate was developed containing the UMP precursor,
05 orotic acid. Adminis~ration of this AsO~-PL-orotic
acid conjugate resulted i~ superior protection of
normal hepatocyte~ in vi~o from higher dose~ of
galactosamine. The addition of an agent that blocks
de no~o synthesis of uridylates, such as
N-phophonacetyl 6-aspar~ate ~PA1A), with galactosa~ine
should increase to~icity to h~patomas. Pretreatment
with th~ orotic acid conjugate (infused over 4 .h)
provided significant protection from PALA plus even
higher do~es of galactosamine (Table 1).
Table I: Ef~ect o~ a Targetable Orotic Acid
Conjugate on Hepatoto~icity and Sur~ival in Rats
Treated ~ith H~patoto~in~
Dose Median Serum Percent
He~totox~ tm~ Pretrea~ent ALT L~el~ Survival
Galactosamine 500Carrier-orotic
acid conjugate 503 100
Saline alone ~,329 100
Carrier alone 2,883 100
Galacto~amine 800~20Carrier-orotic
~PA~A acid conjugate 3,012 100
~-Phosphonacetyl Saline alone >10,000 0
L-Aspartate) Carrier alone 8,382 0
. Orotic acid alone >10,000 0
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WO91~17761 PCT/US91/03291
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E~uivalents
Those skilled in the art will recognize, or be
able to ascertain using no more than routine
experimentation, many equivalents to the specific
05 embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the
following claims.
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