Note: Descriptions are shown in the official language in which they were submitted.
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nESCRTPTION
Sl~ SIT~7,~TION OF ~,R2/ne~c OVh'RF,XPlE~S~SIl~G
C~l~CF',R CF,~,T,~ TO CRli'l\/lOT~,R~Pl~ TIC nl~UG~
BACKGROUNI) O~ THE ~Vl; l~TION
A. Fi.ol-l of the ~nv~ntjon
lQ
The present invention relates to the treatment of cancer. In some aspects, this
invention relates to the suppression of oncogenesis that is merli~te~l by the HER-2/neu
(neu) oncogene, an oncogene which has been correlated with many cancer types in
hllm~n~ Methods and compositions for the treatment of neu-mediated cancer using drug
combinations are disclosed.
B. R~ sl~ 1 of the R~ d Art
Breast cancer remains a major cause of cancer death in women. It is f~stim~ted
2 0 that 180,000 new cases of breast cancer are diagnosed and 46,000 women die of breast
cancer in the United States alone over a period of one year. Hence, there is an urgent
need to develop novel agents breast cancer diagnosis, prognosis prevention and therapy.
Lung cancer is also a major cause of cancer death in the United States (Silverberg
2 5 et aL, 1988). Lung cancers are usually divided into two groups by clinical and biological
criteria: (1) non-small-cell lung cancer (NSCLC~ (Boring et al., 1994), and (2) small-cell
lung cancer (SCLC). Most small-cell lung cancers are sensitive to chemotherapy,
whereas NSCLC usually are refractory to chemotherapy at the time of diagnosis (Minna
et al., 1989). Thus NSCLC are the cause of most lung cancer deaths (Boring, et al.,
1994). To identify more effective th~;ld~t;uLic agents for lung cancer, intensive effort has
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been made to characterize specif1c gene alteration in lung cancers and to develop
therapies that target those genes.
It is well established that a variety of cancers are caused, at least in part, by
genetic abnorrnalities that result in either the over-expression of one or more genes, or
the expression of an abnor~nal or mutant gene or genes. For example, in many cases, the
expression of oncogenes is known to result in the development of cancer. "~ncogenes"
are genetically altered genes whose mut~te~l e~ s~ion product somehow disrupts
normal cellular function or control (Spandidos etal., 1989).
Most oncogenes studied to date have been found to be "activated" as the result of
a mutation, often a point mutation, in the coding region of a normal cellular gene, ie., a
"proto-oncogene", that results in amino acid substitutions in the expressed protein
product. This altered expression product exhibits an abnormal biological fimction that
takes part in the neoplastic process (Travali et al., 1990). The underlying mutations can
arise by various means, such as by chemical mutagenesis or ionizing radiation. Anumber of oncogenes and oncogene families, including ras, myc, neu, raf; erb,src,fms,
jun and abl, have now been identifled and characterized to varying degrees (Travali et
al., 1990; Bishop, 1987).
2 0
About one third of the known oncogenes encode proteins that phosphorylate
tyrosine residues on other proteins. These en~:ymes are known as tyrosine specific protein
kin~es The reaction they catalyze is the transfer of the t~nninS~l phosphoryl group of
ATP to the hydroxyl group of a tyrosine residue in a protein. Included in this family of
2 5 oncogenes are src,yes,fps,fes, abl,ros,fgr,erbB,~ns, mos,raf, and neu.
The neu gene (also known as H~R-2/neu or c-erb-2) encodes a 185-kDa
transmembrane tyrosine kinase (pl85neU) with homology to epidermal growth factorreceptor (Hung et al., lg86; Coussens et al., 1985; Schechter et al., 1984; Semba et al.,
1985; Yamamoto et al., 1986). Enhanced expression of neu is known to be involved in
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many human cancers, including NSCLC and has been shown to correlate with poor
patient survival in NSCLC (Kern et al., 1990, Schneider et al., 1981; Weiner et al.,
1990). Cellular and animal studies have shown that an increase in neu tyrosine kinase
activity increases the ~x~r~s~ion of m~lign~nt phenotypes (Muller et al., 1988; Hudziak
et al., 1987, Muthuswamy et al., 1994; Yu et al., 1991; Yu et al., 1993; Hung ef al.,
1989; Sistonen et aL, 1~89; Yu et al., 1994).
The neu oncogene, was first i~lentified in transfection studies in which NIH 3T3cells were transfected with DNA from chemically induced rat neuroglioblastomas (Shih
0 et al., 1981). The pl85 protein encoded by neu has an extracellular, transmembrane, and
intracellular clom~in, and therefore has a structure consistent with that of a growth factor
receptor (Schechter et al., 1984). The human neu gene was first isolated due to its
homology with v-er~B and EGF-r probes (Semba et al., 1985).
The neu oncogene plays an i~ o~ll role in carcinogenesis. The gene is
arnplified in approximately 30% of primary breast cancer. Amplified expressions of the
neu oncogene in transfected 3T3 cells induces m~ n~nt transforrnation.
Along with an increased proliferative potential, neu-me~ t~c~ cancers appear to
2 0 be resistant to host defense mef~h~ni.~m.~. Studies have shown that overexpression of the
neu oncogene in ~ r~-;Led cells results in r~ t~nce to tumor necrosis factor, a major
effector molecule in macrophage-mt?~ te-l turnor cell cytotoxicity.
neu ex~les:~ion has also been detected in ovarian cancer and its o~ sion
2 5 results in poor prognosis. The ~ s~ion of neu oncogenes in human tumor cells induce
rç~i~t~nre to several host cytotoxic meçh~ni.~m.~.
Therefore, neu oncogene t;~re~ion is correlated with the incidence of cancers ofthe human breast and female genital tract. Moreover, amplification/ove.ex~lt;s~iorl of
this gene has been directly correlated with relapse and survival in hurnan breast cancer
.
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(Slamon et al., 1987; 1989). Therefore, it is important to evolve information regarding
the neu oncogene, particularly in~orrnation that could be applied to reversing or
suppressing the oncogenic progression that seems to be elicited by the presence or
activation of this gene. Unfortunately, little has been previously known about the
manner in which one may proceed to suppress the oncogenic phenotype associated with
the presence of oncogenes such as the neu oncogene.
In addition, neu ovelG~ . ssion in NSCLC is associated with shortened survival.
In vitro experimental models have provided evidence that, in the murine cell NIH 3T3,
oncogenes increase drug resict~n~e. Furthermore, Tsai et al., 1993 and 1995 used a
NSCLC model to den~on~trate that activation of an oncogene is qu~~ ti~ely associated
with intrinsic chemoresi~t~nce in human m~lign~nt cells. This re~i~t~n-~e is observed
with a variety of drugs that are structurally unrelated and act on different targets and/or
by different mech~ni.cm~. Thus increased ~ G~ion of neu oncogene enhances
chemoresistance to a wide variety of chemotherapeutic agents (Tsai, 19933 including
cisplatin, doxorubicin, and VP16 (Tsai et al., 1993; Tsai et al., 1995). The association of
neu O~.G~essiOn in cancer cells with m~lign~nt phenotypes and chemoresict~nçe
provides a plausible interpretation for the poor clinical outcome for patients with
neu-ove~ res~ gtumors.
Although breast cancer diagnosed in its earliest clinical stages (stage 0, stage Ia)
is highly curable, the cure rate for more advanced stages drops precipitously, even after
modern combined-modality treatments. Metastatic breast cancer responds to both
chemotherapy and hormone therapy, and most p~ti(?nt~ can be p~ te~i adequately during
the 1 to 3 years of usual survival. However, metastatic breast cancer is considered
incurable, as demonstrated by the relentless death rates, regardless of the tre~tment
modality l.tili7t~1 Front-line chemotherapy or hormone therapy prograrns for correctly
selected patients produce objective responses in 50% to 70% of patients, but the median
duration of response is usually less than one year. Response rates after second line
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tre~tmer1t~ are considerably lower (20% to 50%), and response durations average 6
months.
Ovarian cancer is also highly curable in its earliest stages, but the overwhelming
ma~ority of patients are diagnosed in stages III and IV. Although responsive to
chemotherapy, most patients with advanced ovarian cancer relapse and die of their
disease. With the introduction of several new cytotoxic agents (t~ n~, vinorelbine,
pl~tinllm derivatives), some responses are observed after second line therapy too, but
cure in this situation remains an elusive goal.
Therefore, there is an urgent need to develop novel anti-cancer agents for thesetypes of cancer.
SUMM~l~Y OF TT~F, Il~IVF,l~TION
The present invention seeks to overcome these and other drawbacks inherent in
the prior art by ~rlministering an agent that sensitizes cancer cells to chemotherapeutic
agents.
2 0 Some embo-lim~nt~ of the invention involve methods of inhibiting transformation
of a cell, in some particular embodiment, oncogene-mediated kansformation of a cell.
Generally, these methods comprise the step of contacting the cell with an emodin-like
tyrosine kinase inhibitor and a chemotherapeutic drug in amounts effective to inhibit the
transformed phenotype. In a ~iefellc,d embodiment, the kansformation being inhibited
will be neu oncogene-mediated tran~r~lnalion. Also, preferably, the embo~;mçn~ in
which transformation is to be inhibited will comprises a tyrosine specific protein kinase
encoded by neu. Of course, the invention also applies to methods of inhibiting other
oncogene-mediated kansformation events, such as transformation by ras, src, yes, J~ps,
fes, abl, ros, fgr, erbB, fms, mos, raJ~, etc.
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Emodin-like tyrosine kinase inhibitors of the invention are those compounds thatexhibit similar characteristics to those of emodin with regard to tyrosine kinase inhibition
and the inhibition of neu-mediated transformation. Typical of such emodin-like tyrosine
kinase inhibitors are anthraquinones that have a chemical structure identical or similar to
those in Table 1. Of course the invention is not limited to the use of these inhibitors and
other inhibitors that possess the structural and/or functional properties of emodin may be
used. In some preferred embofliment~, the emodin-like tyrosine kinase inhibitor is an
anthraquinone tyrosine kinase inhibitor. The emodin-like tyrosine kinase inhibitor may
be, for example, emodin, emodin-8-O-D-glucoside, chrysophanic acid,
gluco-chrysophanic acid, physcion, or physcion-~-O-D-glucoside or any of the other
structures in Table 1 for example, DK-III-8; DK-III-19; DK-III-47; DK-III-48;
DK-III-13; DK-III-11; DK-II-l; DK-II-2; DK-IV-l; DK-V-47; DK-V-48; DK-III-52. Inone preferred embo~iment, the neu tyrosine kinase inhibitor is ~K-V-47. In the most
~lef~ ;d embodiment the neu tyrosine kinase inhibitor is emodin.
In some embo~limçntc of this invention a cell is contacted with between about
O.Smg/kg total weight and 500mg/kg total weight of the emodin-like tyrosine kinase
inhibitor. In some particular embodiments, the cell is contacted with between about
0.5mg/kg total weight and 500mg/kg total weight of emodin. In still other embo~liment~
the cell is contacted with between about 0.5mg/kg total weight and SOOmg/kg total
weight of an emodin-like tyrosine kinase inhibitor. In yet other embodiments the cell is
contacted with between about 10 to about lOO,uM emodin, or bt;lw~en about 20 and 80
,uM, or between about 30 and 7011M, or between about 40 and 60 ,uM or about 50,uM
emodin. . Total weight may be defined as the total weight of the cell or cells in culture,
2 5 or the body weight of an animal, including a human.
Some embo~iiment~ of the invention involve chemothc.d~ uLic agents. These are
compounds that exhibit some form of anti-cancer activity. In some preferred
embodiments, the chemot~.~t;ulic drug is an alkylating agent, plant aLtcaloid, antibiotic,
or antineoplastic agent. In those embodiments of the invention where the
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chemotherapeutic is an alkylating agent, the alkylating agent may be, for example,
mechloreth~min~, cyclophosphamide, ifosfamide chlorambucil, melphalan, bn~lllf~n,
thiotepa, c~rmll~tin~, lormlstin~, and/or shreptozoin. In those embo-1im~nts where the
chemotherapeutic agent comprises a plant alkaloid, the plant alkaloid is, for example,
vincristine, vinblastine or taxol. In a l"~r~,led embodiment, the plant alkaloid is taxol.
In those embodiments of the invention where the chemotherapeutic agent is an antibiotic,
the antibiotic may be, for example, dactinomycin, daunorubicin, idarubicin, bleomycin
mitomycin or doxorubicin. In most preferred embo~iment~ the antibiotic is doxorubicin.
In other embodiments where the chemotherapeutic agent comprises an antineoplastic,
the preferred antineoplastic is, for exarnple, cisplatin, VP l 6 and TNF.
In certain embo-liment~ of the invention, the emodin or emodin-like tyrosine
kinase inhibitor is ~(lmini~tered to the cell prior to the ~rlmini~tration of the
chemotherapeutic agent. In other aspects of the invention, the chemotherapeutic agent is
~lminictered to the cell prior to z~flmini~tration of the emodin or emodin-like inhibitor.
Alternatively the emodin or emodin-like tyrosine kinase inhibitor and the
chemoth~,.d~uLic drug are ~flmini~tered simnlt~neously.
In some embo-liment~ of the invention, the cell is located within an animal and
2 0 effective amounts of the emodin or emodin-like tyrosine kinase inhibitor and the
chemotherapeutic drug are ~lmini~tered to the animal. ln certain embodiments of the
invention, the chemotherapeutic drug and the emodin or emodin-like tyrosine kinase
inhibitor are suitably dispersed in a ph~ cologically acceptable formulation. In certain
preferred embodilllelll~, where the cell is an animal cell, the cell is a hDan cell. In other
preferred embodiments the cell is a lung, cancer cell, ovarian cancer cell, or a breast
cancer cell.
In some embo~lim~nt~ of the present invention the cell is c-)nt~cte~l with a single
composition compricin~ emodin or an emodin-like tyrosine kinase inhibitor in
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combination with a chemotherapeutic agent. In such cases, the composition may besuitably dispersed in a pharmacologically acceptable formulation.
The invention contemplates embo-1iment~ comprising sç~iti7inp a cancer cell to
a chemotherapeutic drug. These emborliments comprise exposing the cell with an
effective amount of emodin or emodin-like. In some such embo~liment~ inhibition of
neu-m~ te~1 cancer is accomplished by ~-lmini~trating an effective combination of
emodin-like tyrosine kinase inhibitor and chemotherapeutic drug to an animal having or
suspected of having cancer in an effective amount to inhibit the cancer. In embodiments
where the composition is ~llmini~tered to an animal, the animal is typically a m~n m7~l
In such cases, the invention will be of particular use in the kç~tment and prevention of
neu-m~ ted transformation in hlln~n~
Certain embo~im~nt~ of the present invention comprise injecting a therapeutically
effective amount of an emodin-like tyrosine kinase inhibitor into an animal and
contacting the animal with a chemotherapeutic drug. In preferred embo-lim~nt~ the
emodin-like tyrosine kinase inhibitor is an emodin. In certain embodiments of the
invention a cancer site is contacted with a chemotherapeutic drug by ~lnnini~tering to the
animal a therapeutically effective amount of a ph~rm~t~eutical composition comprising a
chemotherapeutic drug wherein the chemotherapeutic drug is for example cisplatin,
doxorubicin, VP 16, taxol or TNF.
The invel~lols have also enabled the production of ph~rm~celltical compositions
comprising an emodin-like tyrosine kinase inhibitor and a chemothe.dpeulic drug in a
2 5 ph~ cological carrier. Those of skill will understand the nature of such
ph~rm~cological carriers based on the te~cltings of this specification and t_e current
knowledge in the art. The pharm~ce~ltical culn~o~i~;ons of the invention may contain
any of the emodin-like tyrosine kinase inhibitors and chemotherapeutic drugs mentioned
above or elsewhere in this specification, or know to those of skill in the art. In a
3 0 ~ Ç~ d ph~ eutical composition the chemotherapeutic drug is cisplatin,
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doxorubicin, etoposide, taxol or lNF. In some preferred embo~lim~ntc, the emodin-like
tyrosine kinase inhibitor is emodin.
The invention also encomp~es ph~rrn~ceutical combinations comprising an
emodin-like tyrosine kinase inhibitor and a chemotherapeutic drug. In certain plcrc.led
combinations, the tyrosine kinase inhibitor is emodin. The chemotherapeutic drug may
be any that is listed elsewhere in this specification or known to those of skill in the art at
the present or in the future. Exemplary chemotherapeutic drugs fi~r us in the
pharmaceutical combinations o~ the present invention are cisplatin, doxorubicin,etoposide, taxol and TNF. In certain embodiments of the invention the ph~rm~ceulical
combination may contain the emodin or emodin-like tyrosine kinase inhibitor and the
chemoth~,.d~t;uLic drug within the same ph~nn:~ceutical composition. In other
embo~limçnt~, the ph~ ceutical combinations will comprise separate ph~rn~reutical
compositions for each of the emodin or emodin-like tyrosine kinase inhibitor and the
chemotherapeutic drug. These separate compositions may be combined int~rn~l to or
e~ctt?rn~l to a body to create the ph~rrn~t~eutical combination.
Other embodiment~ of the invention include therapeutic kits comprising in
suitable container, a ph~rmz~ceutical formulation of an emodin-like tyrosine kinase
2 0 inhibitor, a ph~ reutical formulation of a chemotherapeutic drug, and/or a
ph~ relltical formulation comprising both emodin or an emodin-like compound and a
chemotherapeutic drug. The kit may also contain instructions on how to a-lrninict~r the
h~rrn~relltical formulation or formulations of the kit to an animal either alone, or in
combination with formulations that one may obtain separately from the kit. The kit may
also comprise instructions that explain how to use the kit but are provided separately
from the container of the kit. The kit may comprise the emodin or emodin-like tyrosine
kinase inhibitor and chemothe~ Lic drug to be present within a single container or
alternatively the kit could comprise the emodin-like tyrosine kinase inhibitor and/or the
chemotherapeutic drug are present within distinct cont~in~r.c
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Some embodiments of the present invention relate to a method of inhibiting
oncogene-mef1i~te-~ transformation of a cell, comprising cont~tin~ the cell with emodin
or an emodin-like tyrosine kinase inhibitor. These compounds are well-described in this
specification.. In ~I~;r~l.ed embo~1iment~ the oncogene-mediated l~ r~ lation is neu
oncogene-mediated transformation. Also, preferably, the embo-liment~ in which
transformation is to be inhibited will comprises a tyrosine specific protein kinase
encoded by neu. The invention also contemplates ph~rm~ eutical compositions, and kits
comprising emodin or emodin-like tyrosine kinase inhibitors to inhibit neu-me~ te~l
transformation. Of course, the invention also applies to methods of inhibiting other
oncogene-me~i~t~-1 transformation events, such as transformation by ras, src, yes,~s,
fes, abl, ros, fgr, erbB, fins, mos, raf:
RRT~F nl~ CRTPIION OF T~ r)E~WIl~S
The following drawings form part of the present specification and are included to
further demonstrate certain aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein.
2 0 FIG. 1A and FIG. lB. Effect of dose dependence and time course of emodin
tre~tm~nt on tyrosine phosphorylation and expression of neu in human breast cancer
MDA-MB453 cells. FIG. 1 A, cells in the serum-free medium were incubated without(O) or with emodin (10 or 40 ,uM) at 37~C for 24 h. FIG. lB, cells were incllh~te~1
without (-) tre~tment or with (+) emodin (40 ~M) at 37~C for dirr~c;nl times. Cell
2 5 extracts were immlmnprecipitated by anti-pl 85neU antibody (Anti-neu) and then w~
blotted with either antiphosphotyrosine antibody ~Anti-PY) or anti-p 1 85neU antibody as
described in ~xample 1.
FIG. 2A and FIG. 2B. Effect of emodin on tyrosine phosphorylation and
expression of neu in human neu overexpressing breast cancer cells. Cells in the
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serum-free medium were incubated without (-) or with (+) 40 ~lM emodin at 37~C for 24
h, and then cell Iysates were immIlnoprecipitated by anti-pl85neU antibody and blotted
with anti-phosphotyrosine (FIG. 2A) or with anti-p 1 85neU (FIG. 2B) antibodies as
described in Example 1.
FIG. 3A and FIG. 3B. Effect of emodin on autophosphorylation and
transphosphorylation of P18~neU in MDA-MB453 breast cancer cells. FIG. 3A show
cells that were inc~ t~A without (lane 1) or with (lane 2) emodin (40 ,uM~ at 37~C for
24 h, then cell lysates (500 ,ug) were imrnunoprecipitated and kinase activities were
measured by incubation with [~-32P]ATP and enolase. Cell Iysates from untreated cells
were imm~lnoprecipitated, then incubated with [~-32P]ATP, enolase and different
concentrations of emodin for 20 minutes at room t~ re (FIG. 3B). I~e~çt~nt.~ were
resolved on 7.5% SDS-PAGE. The phosphorylation products were dried and visualized
by autoradiography as described in Example 1.
FIG. 4A and FIG. 4B. Ef~ect of emodin on the proliferation of human breast
cancer cells expressing different levels of neu. MDA-MB453, AU-565, BT-483 cells,
which overexpress neu and MCF-7, MDA-MB23 1, HBL- 100 cells, which express
normal levels of neu, were incubated without or with different concentrations of emodin
2 0 at 37~C for 72 h (FIG. 4A). The effect on cell growth was çx~mine(l by MTT assay, and
the percentage of cell proliferation was calculated by defining the absorption of cells
without treatment of emodin as 100%. MDA-MB453 cells were incub~tt?cl without
(control) or with emodin (40 ,uM) at 37~C for different times, and cells were washed and
counted by trypan blue exclusion with hemacytometer (FIG. 4B). All detl,~.,,i.,;.lions
2 5 were made in triplicate. Results are mean + SD.
FIG. 5. Effect of emodin on human breast cancer cell colony growth in soft
agarose. Cells (1 x 103 cells/well) were seeded into 24-well plates in culture medium
cont~ining 0.35% agarose over a 0.7% agarose layer with or without 40 ~lM emodin, and
3 0 incubated for 3 weeks at 37~C. Colonies were stained with p-iodonitrotetrazoliurn v;olet
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and counted the percentage of colony ~ormation was calculated by defining number of
colonies in the absence of emodin as 100%. All deterrninations were made four times.
Results are mean + SD.
FIG. 6A and FIG. 6B. The effect of emodin on tyrosine phosphorylation and
expression of neu in human lung cancer cells. FIG. 6A and FIG. 6B shows the effect
of emodin on tyrosine phosphorylation and expression of neu in hurnan lung cancer cells.
Cells were grown in serum-free medium were incllk~tecl without (-) or with (+) 30 ~lM
emodin at 37~C for 24 hours, and then cell Iysates were separated by 6.5% SDS-PAGE
and blotted with anti-phosphotyrosine ~anti-PY) or with anti-pl85ne" (anti-neu)
antibodies as described in Example l. FIG. 6A shows the effects of 30 ,uM emodin on
tyrosine phosphorylation in NCI-H1435 and NCI-H226. FIG. 6B shows the effects of30 IlM emodin on tyrosine phosphorylation in NCI-H460, H460.neo, H460eB2 and
H460eB3 cells
FIG. 7A and FIG. 7B. Effect of emodin on the proliferation of human lung
cancer cells. Hurnan lung cancer cells were incubated with or without different
concentrations of emodin at 37~C for 72 hours. The effect on cell growth was çx~-nin~cl
by MTT assay, and the percentage of cell proliferation was calculated by defining the
2 0 absorptiorl of cells without treatrnent of emodin at 100%. All ~lefennin~tions were made
in six replicates. Results are means + SD. FIG. 7A shows the effects of emodin on the
proliferation of NCI-H1435 and NCI-H226 as measured by MTT assay. FIG. 7B shows
the ef~ects of emodin on the proliferation of NCI-H460, H460 neo, H460eB2 and
H460eB3 as measured by MTT assay.
FIG. 8A, FIG. 8B and FIG. 8C. Effects of cisplatin, doxorubicin, and VP16
alone or in combination with emodin on the proliferation of human lung cancer
cells. The effects on cell growth of NCI-H1435, HCI-H226, HCI-H460, H460.neo,
H460.eB2 and H460.eB3 cells were examined by MTT assay and the drug concentrations
3 0 required to inhibit 50% of cell growth (IC50) were calculated. Cells were incubated with
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the drugs at 37~C for 72 h. FIG. 8A shows the IC50 for cisplatin in NCI-H14-35,
HCI-H226, HCI-H460, H460.neo, H460.eB2 and H460.eB3 cells. FIG. 8B shows the
IC50 for doxorubicin in NCI-H1435, HCI-H226, HCI-~460, H460.neo, H460.eB2 and
H460.eB3 cells. FIG. 8C shows the IC50 for VP16 in NCI-H1435, HCI-H226,
HCI-H460, H460.neo, H460.eB2 and H460.eB3 cells.
FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G and FIG.
9H. The effects of drug combinations on cell growth. All cells were treated with 30
~LM emodin alone or in combination with different drugs at 37~C for 72 h. The effect on
cell growth were e~ in~d by MTT assay, and the percentage of cell proliferation was
calculated by defining the absorption of cells not treated with emodin as 100%. The
inhibitory effect was calculated by 100% minus percentage of cell proliferation. All
determin~tions were made in six replicates. Results are means + SD.* Indicates
synergism which were evaluated as described in Example 1. FIG. 9A shows the effects
of drug treatment on NCI-H1435 cells these are 50 ,uM cisplatin, l.0 IlM doxorubicin or
1.0 ,uM VP16. FIG. 9B shows the effects of drug tre~tm~nt on NCI-H226 cells. These
cells were treated with S ~lM cisplatin, 0.5 IlM doxorubicin, or 0.1 tuM VP16. FIG. 9C
shows the effects of drug treatment on NCI-H460. These cells were treated with 5 ,uM
cisplatin~ 0.1 ~LM doxorubicin, or 0.1 ~M VP16. FIG. 9D shows the effects o~ drug
2 0 treatment on H460.neo. These cells were treated with 5 IlM cisplatin, 0.1 ~LM
doxorubicin, or 0.1 ~LM VP16. FIG. 9E shows the effects of drug treatment on
H460.eB2. These cells were treated with 75 ,uM cisplatin, 0.5 ~LM doxorubicin, or 0.5
~lM VP16. FIG. 9F shows the effects of drug tre~tment on H460.eB3 These cells were
treated with 5 ~M cisplatin, 0.5 ,uM doxorubicin, or 0.5 ~lM VP16. FIG. 9G. shows the
effects of emodin, taxol and TNFa on MDA-MB361 human breast cancer cells. These
cells were treated with 20,uM emodin alone or in combination with lOnM taxol andO.SnM TNF-a. FIG. 9~I. shows the effects of emodin, taxol and TNF-a on
MDA-MB435 human breast cancer cells. These cells were treated with 20~1M emodin
alone or in combination with 0.1 nM taxol and 0.1 nM TNF-a.
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FIG. lOA, FIG. 10B, FIG. lOC, FIG lOD, FIG. lOE AND FIG 101~. The
effects of drug combinations on cell growth in soft agar. Cells (lx103 cells/well) were
seeded into 24-well plates in culture medium cont~inin~ 0.35% agarose over a 0.7%
agarose layer. Colonies were stained with p-iodo~ ot~Ll~olium violet and counted the
percentage of colony formation was calculated by defining nurnber of colonies in the
absence of emodin as 100%. All determinations were made four times. Results are
mean ~ SD. FIG. lOA shows the effects of 30 ,uM emodin alone or in combination with
S ~LM cisplatin, 0.1 ~LM doxorubicin or 0.1 ,uM VP16 on the cell growth of NCI-H460
cells. FIG. 10B shows the effect of 30 ~LM emodin alone or in combination with 5~M
cisplatin, 0.1 ,uM doxorubicin or 0.1 ,uM VP16 on the cell growth of NCI-H460.neo cells
in soft agar. FIG. 10C shows the effect of 30 ~M emodin alone or in combination with
75 ~M cisplatin, 0.5 ,uM doxorubicin or 0.5 ~LM VP16 on the cell growth of H460.eB2
cells in soft agar. FIG. 10D shows the effect of 30 ,uM emodin alone or in combination
with 75 ~LM cisplatin, 0.5 ~M doxorubicin or 0.5 ~LM VP16 on the cell growth of
H460.eB3 cells in soft agar. FIG. lOlE shows the effect of 30 ,uM emodin alone or in
combination with S IlM cisplatin, 0.1 ~M doxorubicin or 0.1 ,uM VP16 on the cellgrowth of NCI-H226 cells in soft agar. FIG. lOF shows the effect of 30 ,u~ emodin
alone or in combination with 50 ~M cisplatin, 1 ~LM doxorubicin or 1 ,uM VP16 on the
cell growth of NCI-H1435 cells in soft agar.
FIG. 11. Effect of emodin on colony growth of human breast cancer cells (A)
and activated HER-2/neu transforme(} 3T3 cells (B) in soft agarose. Cells (1 x 103
cells/well) were seeded into 24-well plates in culture medium cont~inin~ 0.35% agarose
over a 0.7~/0 agarose layer with or without dirr~ l concentrations of emodin or
2 5 DK-V-47, and incubated for 3 weeks at 37~C. Colonies were stained with
p-iodonitrotetrazoliurn violet and counted the percentage of colony forrnation was
calculated by defining number of colonies in the absence of emodin and DK-V-47 as
100%. All d~le~ tions were made four times. Results are mean ~ SD.
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FIG. 12. Effect of emodin and DK-V-47 on autophosphorylation and
transphosphorylation of pl85neU in activated HER-2/neu transformed 3T3 cells.
~ Cell lysates from untreated cells were immnnoprecipitated, then incubated with
[~-32P]ATP, enolase and different concentrations of emodin and DK-V-47 for 20 min at
room t~ pel~L~lre. ~e~ctAnt~ were resolved on 7.5% SDS-PAGE. The phosphorylationproducts were dried and visualized by autoradiography.
FIG. 13. Effect of emodin on the proliferation of activated HER~ eu
transformed 31['3 cells. Cells were incubated without or with different concentrations of
emodin or DK-V47 at37~C for 72 h. The effect on cell growth was ex~mine~l by MTTassay, and the percentage of cell proliferation was calculated by defining the absorption
of cells without tre~tment of emodin and DK-V-47 as 100%. All detPrmin~tions were
made in triplicate. Results are mean + SD.
FIG. 14. Emodin and DK-V-47 reduce gelatinolytic activity of activated
HER-2/neu-transformed 3T3 cells. Cells were treated or not treated with varying
concentrations of emodin and DK-V-47 overnight, the culture supern~t~n~ were
collected and analyzed by zymography using gelatin-embedded SDS-PA~3E.
Gelatinolytic enzymes were detected as transparent bands on the blue-background of
2 0 Coomassie blue-stained gels. Positions of the 92-kDa and 68-kDa gel~tin~e are
indicated.
FIG. 15. Emodin and DK-V-47 inhibit invasion of a~ alt~d
HER-2/neu-transformed 3T3 cells that have in~ DE-I tyrosine kinase a~liYiL~
through a Matrigel layer. In vitro invasion was measured by using 24-well transwell
units with an 8-mm pore polycarbonate filter coated with Matrigel to form a thin,
continuous layer on the filter top. The lower co~ llent contained 0.6 ml of l~minin
(20 mg/ml) as chemo-attractants. Activated ~ER-2/neu-transformed cells (5 x 104) were
placed in the upper compartment and treated or not treated with emodin or DK-V-47 for
3 days. Then, lower surfaces of filters from the transwell units were fixed with 3%
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glutaraldehyde in PBS and stained with Giemsa solution. Chemo-invasive activity was
deterrnined under the microscope by counting cells that had migr~te-i to the lower side of
the filter. All ex~ nents were done in triplicate.
FIG. 16A and FIG. 16B. Effect of taxol alone or in combination with emodin on
the human breast cancer cell colony growth in soft agarose. Cells (1 x 103 cells/well)
were seeded into 24-well plates in culture medium co..l;~ .g o.35% agarose over a 0.7%
agarose layer with or without either taxol or emodin alone, or emodin plus taxol and
incubated for 3 weeks at 37~C. All cells were treated with 20 ,uM emodin alone or in
combination with taxol. The doses of taxol used to treated different cancer cells are 1 ,uM
for MDA-MB 361 cells (FIG. 16A); 0.l nM for MDA-MB 435 cells (FIG. 16B).
Colonies were stained with p-iodonilr~ zolium violet and counted, and the percentage
of colony formation was calculated by defining the number of colonies in the absence of
drugs as 100%. All determin~tions were made four times. Bars, SD.
FIG. 17A and FIG. 17B. FIG. 1 7A. Tumor growth in mice bearing
HER-2/neu-owlcx~lessillg breast cancer (n - l0). HER-2/neu-overexpressing
MDA-MB361 cells (5 x 107) were injected into female nu/nu mice subcutaneously.
Three weeks later, when the palpable solid tumors were detecte-i The mice were give
either placebo, emodin (40 mg/kg body weight) or Taxol (l0 mg/kg body weight), or
Emodin (40 mgA~g) plus taxol (l0 mg/kg) by i.p. injection twice a week for 8 weeks. The
tumor volume was monitored weekly for 8 weeks. Mice were observed for survival for
up to 300 days. FIG. 17B. Survival in mice bearing H~R-~/neu-ov~lcx~l.,ssing breast
cancer.
r~FTATT,Fn DF~CRTPTION T~F PRFFli,RRFn Fl~Ol~Il~,l~T.~
Ihe present invention seeks to overcome drawbacks inherent in the prior art by
providing new tre~tment methods, compositions and kits for increasing the efficacy of
3 0 antineoplastic agents against cancer
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The invention provides methods for treating cancers using emodin and/or
, emodin-like compounds that inhibit neu-tyrosine kinase activity. The methods of the
invention generally rest in using emodin and/or emodin-like compound alone or incombination with another anti-cancer agent effective to treat the cancer cells.
In one preferTed embo-liment, the inventors have discovered that the use of
emodin, an exemplary emodin-like tyrosine kinase inhibitor, in combination with
antineoplastic agents has a synergistic effect in inhibiting the growth of neu-mediated
cancers, which are usually chemoresistaIlt.
The methods, compositions and kits of the invention may be used in conJunclion
with any emodin-like drug that has an active species or metabolite that, at least in plart,
inhibits protein tyrosine kinase activity. Such emodin-like inhibitors may themselves be
the "active species". Emodin, chrysophanic acid, physcion and the glucosides of these
compounds amongst others, are examples of this group of emodin-like inhibitor
analogues. Many emodin-like inhibitors are, like emodin, anthraquinones. Alternatively,
the emodin-like compound or drug may be one that exhibits similar properties to emodin
in relation to neu-mediated cancer or is metabolized within the body to provide an active
2 0 species or metabolite.
Structural Properties of F.mo~in and Anthraquinone-Based Emodin-Like
Compounds
Emodin (3-methyl-1, 6, 8 trihydroxyanthra-quinone) belongs to a group of
compounds that are structurally based upon the structure of anthraquinone shown
in Table 1, to which various R groups may be added. A wide variety of anthraquinones
exist in nature (Yeh et al., 1988; Kupchan and Karim, 1976; Jayasuriya et al., 1992). .
Structure B in Table 1 is emodin itself; C is emodin-8-0-D-glucoside; D is chrysophanic
- 30 acid; E is gluco-chrysophanic acid; F is physcion, and G is physcion-8-0-D-glucoside.
The emodin-like compounds of structures A, C and D-G are only exemplary forms of
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emodin-like compounds that may be used in the present invention. Nurnerous otheremodin analogues are available as shown in Table 1 and as described by, e.g., Yeh et al.,
1988; Kupchan and Karim, 1976; Jayasuriya et al., 1992. Further the inventors have
investigated additional emodin-like compounds recently developed by the research group
of Ching-Jer Chang at Purdue University, West Laf~ and initial indications are that
these compounds perform as well as or even better than emodin itself.
The first group (group A: Table I and Table 2~ is comprised of compounds which
structurally related to emodin, only replace CH3 group with different other group at C3 of
emodin, their inhibitory activities of tyrosine phosphorylation of pl85neU are in
following order CH3 > C=NOCH3 > CHNOH > CH20H > CONH2 > COOH and
inhibitory activities for proliferation of cells are CH3 > CHNOH > CONH2 > C=NOCH3
> CH20H > C~20H > COOH, these results indicate that CH3 group at C3 position of
emodin is very important to remain inhibitory activities of emodin on tyrosine
phosphorylation and proliferation.
The second group (Group B: Table 1 and Table 2) also structurally related to
emodin, only replace OH group with either H or OCH3 group at C6 position of emodin.
However, compare with emodin, their inhibitory activity for both tyrosine
2 0 phosphorylation of pl 85neU and proliferation of cells are 5-fold lower than emodin.
The third (Group C), after removed OH groups at C~, C6 and C8, and CH3 group
at C3 of emodin, added NH2 group at Cl and C2 of emodin, decrease activity of emodin.
The fifth group (group ~ remove ketone group from C 10, also reduce activity of
2 5 emodin.
The fourth group (group D), structurally similar to the third group, except replace
Cg ketone with either p-acetylamidebenzomethyl group (DK-V-47) or
p-aminobenzomethyl group (DK-V-48~, DK-V-47 has higher activity than emodin to
inhibit tyrosine phosphorylation of p~85neU and proliferation of cancer cells, however,
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replace COCH3 of DK-V-47 with H group (DK-V-48), DK-V-48 decrease the activity of
DK-V-47. These results suggest that COCH3 group of DK-V-47 is hlll~oll~t to keep the
activity of DK-V47.
TABLE 1
Stl u~Lu~ ~s of Emodin-like Co~ o
~3 A. ANTHRAQUINONE
OH O OH
CH ~ OH
HO o OH
H3C)~ ~ D ~ Jlla,li~, acid
HO O O-~ glucose
H3C~ ~ a d
HO O OH
b~ F. physcion
H3C~ ~I ' ~OCH
OH O glucose
H3C/ ~ ~ G physcion-8-0-Dglucoside
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TABLE 1, Corlti~ r-i
Cu.,.,u~ , R
OH O OH
7~2 H DK-111-8 -CH2OH
~\ ~10~ I DK-111-19 -CONH2
OH ~ lr 4 R J DK-111~7 -C=NOCH3
K DK-111~8 -CNOH
L DK-111-13 -COH
(H-M) M DK-111-11 -COOH
Compounds R'
OH O OH
R~ CH3 N DK-11-1 -H
O DK-11-2 -OCH3
(N-O)
OH O OH
0 ~ ~5 1: J CH3
(P)
O NH Com,uounds R
~1H
H R'
R DK V ~7 -CH~CO
S DK.V 4~ H
l~
(R. 8~
F--nrti~ Properties of h'.mc-lin and ~mo~lin-Like Compounds
Emodin, which was first isolated from polygonum cuspidatum, has been shown to
5be an inhibitor of the protein tyrosine kinase p56lck (Jayasuriya et al.; 1992). In the
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present invention, emodin is shown to inhibit neu tyrosine kinase activity and to
preferentially repress the transformation ability and growth rate of neu-ovel~x~l~ssing
breast cancer cells. Emodin has been reported to be a tyrosine kinase inhibitor that
restricts the activity of p561ck kinase by preventing the binding of ATP in vitro
(Jayasuriya et al., 1992). Emodin a}so can inhibit the growth of cancer cells, including
lymphocytic leukemia (Kupchan et al., 1976), HL-60 human leukemia cells (Yeh et al.,
1988), and ras-transformed human bronchial epithelial cells (Chan et al., 1993), by an
unknown me~h~ni~m
The inventors have found that emodin and emodin-like compounds assist in
overcoming the chemoresistance of neu-overexpressing cancer cells by sen~iti7inp such
cells to chemotherapeutic agents. Having ex~rnint?A the effects of emodin on the tyrosine
phosphorylation, proliferation and morphology, the inventors have e~c~min~l1 the effect
of emodin on tyrosine phosphorylation of neu in cancer cells, and tested the effects of
combinations of emodin with chemotherapeutic agents on proliferation of these cells.
The inventors found that emodin suppressed tyrosine phosphorylation of neu,
preferentially inhibited proliferation of neu-expressing lung cancer cells to a surprising
level, and sensitized these cells to chemotherapeutic drugs. This suppression of tyrosine
phosphorylation is a functional characteristic of emodin-like compounds.
The inventors have demonstrated that emodin and emodin-like compounds
suppress the tyrosine kinase activity of neu-ov~.e~lc;ssing human breast cancer cells,
es~es their transforming ability, and ;n~ s their differentiation. Further, the
inventors find that emodin also ~ e~ses tyrosine phosphorylation of neu in l mg
cancer cells and preferentially inhibits growth of these cells. Further, this invention
demonstrates that emodin is able to sensitize lung cancer cells that o~ ,ess neu to the
chemotherapeutic agents cisplatin, doxorubicin, and VP16. These results suggest that the
tyrosine kinase activity of pl85neU is required for the chemoresistant phenotype of neu
ove.~ essing cancer cells. Therefore, the invention shows that adding emodin to
3 0 chemotherapeutic regimens greatly improves their efficacy.
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Thel a~eulically Err~Liv~ Amounts of Emodin and Emodin-Like Compounds
A therapeutically effective amount of an emodin-like tyrosine kinase inhibitor
that is combined with a second agent as treatment varies depending upon the host treated
and the particular mode of ~lmini~tration. In one embodiment of the invention the dose
range of an emodin-like tyrosine kinase inhibitor used will be about 0.5mg/kg body
weight to about SOOmg/kg body weight. The term "body weight" is applicable when an
animal is being treated. When isolated cells are being treated, "body weight" as used
herein should read to mean "total cell weight". The term "total weight may be~used to
apply to both isolated cell and animal treatment. All concentrations and tre~tment levels
are expressed as "body weight" or simply "kg" in this application are also considered to
cover the analogous "total cell weight" and "total weight" concentrations. However,
those of skill will recognize the utility of a variety of dosage range, for example, lmg/kg
body weight to 450mg/kg body weight, 2mg/kg body weight to 400mg/kg body weight,3mg/kg body weight to 350mg/kg body weight, 4mg/kg body weight to 300mg/kg body
weight, Smg/kg body weight to 250mg/kg body weight, 6mg/kg body weight to
200mg/kg body weight, 7mg/kg body weight to 150mg/kg body weight, 8mg/kg body
weight to lOOmg/kg body weight, or 9mg/kg body weight to 50mg/kg body weight.
Further, those of skill will recognize that a variety of dirrelcllt dosage levels will be of
use, for example, lmg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 7.5mg/kg, 10, mg/kg,
12.5mg/kg, 15mg/kg, 17.5mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 45
mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg, 90mg/kg, lOOmg/kg, 120mg/kg,
140mg/kg, 150mg/kg, 160mg/kg, 180mg/kg, 200mg/kg, 225 mg/kg, 250mg/kg,
2 5 275mg/kg, 300mg/kg, 325mg/kg, 350mg/kg, 375mg/kg, 400mg/kg, 450mg/kg,
500mg/kg, 550mg/kg, 600mg/kg, 700mg/kg, 750mg/kg, 800mg/kg, 900mg/kg,
lOOOmg/kg, 1250mg/kg, lSOOmg/kg, 1750mg/kg, 2000mg/kg, 2500mg/kg, andlor
3000mg/lcg. Of course, all of these dosages are exemplary, and any dosage in-between
these points is also expected to be of use in the invention, as are any ranges of dose
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defined by ant two of these points. Any of the above dosage ranges or dosage levels may
be employed for emodin alone or for emodin in combination with an anti-cancer drug.
-
"Therapeutically effective amounts" are those amounts effective to produce
beneficial results in the recipient animal or patient. Such amounts may be initially
deterrnined by reviewing the published lil~ , by con~ ctin~ in vitro tests or byconducting metabolic studies in healthy experimental ~nim~l~ Before use in a clinical
seKing, it may be beneficial to conduct confirm~tory studies in an animal model,preferably a widely accepted animal model of the particular disease to be treated.
Preferred animal models for use in certain embo~1im~nt~ are rodent models, which are
preferred because they are economical to use and, particularly, because the results gained
are widely accepted as predictive of clinical value.
As is well known in the art, a specific dose level of active compounds such as
emodin and emodin-like compounds for any particular patient depends upon a variety of
factors includin~ the activity of the specific compound employed, the age, body weight,
general health, sex, diet, time of ~-lmini~tration, route of ~(lmini~tration, rate of excretion,
drug combination, and the severity of the particular disease undergoing therapy. The
person responsible for al1mini~tr~tion will, ~iet~rmine the a~ opli~le dose for the indiviclual
2 0 subject. Moreover, for hurnan ~(lmini~tion, pl~lions should meet sterility,
pyrogenicity, general safety and purity standards as required by FDA Office of Biologics
standards.
A composition of the present invention is typically ~-imin;~tered orally or
~c.~ dlly in dosage unit forrnulations co.. l~;.. ;.~g standard, well known non-toxic
physiologically acceptable carriers, adjuvants, and vehicles as desired. The term parental
as used herein includes subcutaneous iniections, intravenous, intr~muccular~ intra-arterial
injection, or infusion techniques.
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In some embodiments, the emodin or emodin-like compound will be
1mini.ct~ed in combination with a second agent. So long as a dose of second agent that
does not exceed previously quoted toxicity levels is not required, the effective amounts
of the second agents may simply be defmed as those amounts effective to reduce the
cancer growth when ~rlmini~tered to an animal in combination with the emodin-like
agents. This is easily determined by monitoring the animal or patient and measuring
those physical and biochemical parameters of health and disease that are indicative of the
success of a given tre~tm~nt Such methods are routine in animal testing and clinical
practice.
Examples of second agents that may be used with emodin or emodin-like tyrosine
kinase inhibitor are anti-neoplastic agents. Examples of these are cisplatin; doxorubicin
(Mechetner & Roninson, 1992) and analogues, such as 14-0-hemiesters of doxorubicin;
etoposide; vincristine ~Shirai et al., 1994, Friche et al., 1993); vinblastine (Bear, 1994;
McKinney & Hosford, 1993); actinomycin D (McKinney & Hosford, 1993);
daunomycin (Bear, 1994); daunorubicin (Muller et al., 1994); taxotere (Hunter e~ al.,
1993); taxol (Mechetner & Roninson, 1992); and tamoxifen (Trump et al., 1992). The
skilled artisan is directed to "Physicians Desk Reference" 15th Edition, for dose ranges of
chemotherapeutic agents practiced in the art. Some variation in dosage will necessarily
2 0 occur depending on the condition of the subject being keated.
The tre~tm~nt methods generally comprise ~rlmini~t~rinE to an animal with
cancer, including a human patient, a therapeutically effective combination of emodin and
or emodin-like tyrosine kinase inhibitor alone or in combination with one or more second
2 5 agents that is effective in keating neu-me~ t.-~l cancer growth exemplified by a decrease
in the activity of neu-protein tyrosine kinase which is over-expressed in neu-m~ te~1
cancers. The second agent(s) may be any of those listed above, and their functional
equivalents.
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Assays for A-~rlition~l Emodin-Like Tyrosine Kinase Inhibitors for Use in the
Invention
In certain embodiments, the present invention concerns a method for identifying
further neu protein tyrosine kinase inhibitors, which are "emodin-like compounds." It is
contemplated that this screening technique will prove useful in the general identification
of any compound that will serve the purpose of in-hibiting neu-protein tyrosine kinase in
a manner similar to the exemplary emodin-like tyrosine kinase inhibitors.
Useful compounds in this regard will not be limited to emodin. In fact~ it may
prove to be the case that the most useful phz~ ological compounds for identification
through application of the screening assay will be compounds that are structurally related
to emodin. The active compounds may include fragments or parts of naturally-occurring
compounds or may be only found as active combinations of known compounds which
are otherwise inactive. However, prior to testing of such compounds in humans oranimal models, it will possibly be necessary to test a variety of c~ndidates to cleterrnine
which have potential.
Accordingly, in screening assays to identify ph~ eutical agents which inhibit
2 0 neu-protein tyrosine kinase activity, it is proposed that compounds isolated from natural
sources, such as ~nim~, bacteria, fungi, plant sources, including leaves and bark, and
marine samples may be assayed as candidates for the presence of potentially useful
rh~nn~-eutical agents. It will be understood that the ph~ elltical agents to be
screened could also be derived or synth~ l from chemical compositions or man-made
2 5 compounds.
In these embodiments, the present invention is directed to a method for
~ t~rminin~ the ability of a c~n~ te substance to inhibit a tyrosine kinase assay, the
method including generally the steps of:
.
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(a) obtaining an enzyme composition comprising a tyrosine kinase, preferably
neu-tyrosine kinase that is capable of phosphorylating tyrosine;
(b) ~lmi~rin~ a candidate substance with the enzyme composition; and
(c) ~letPrminin~ the ability of the candidate substance to inhibit tyrosine
phosphorylation.
To identify a candidate substance as being capable of inhibiting protein
phosphorylation, one would f1rst obtain an enzyme composition that is capable ofphosphorylating tyrosine residues on a protein of interest. Naturally, one would measure
or determine the phosphorylation activity of the tyrosine kinase composition in the
absence of the added ç~n~ te substance. One would then add the candidate substance
to the tyrosine kinase composition and re-(letermine the ability of the tyrosine kinase
composition to phosphorylate tyrosine residues on a test protein in the presence of the
candidate substance. A candidate substance which reduces the phosphorylation activity
of the tyrosine kinase composition relative to the activity in its absence is indicative of a
candidate substance with inhibitor capability.
2 0 The czln~ te screening assay is quite simple to set up and perform~ and is related
in many ways to the assay discussed above for det~rminin~ enzyme activity. Thus, after
obtaining a relatively purified ~ ~dlion of the enzyme, either from native or
recombinant sources, one will admix a ~ ~ntliclzlte s~bst~nce with the enzyme p~ ~dtion,
under conditions which would allow the enzyme to perforrn its tyrosine phosphorylation
2 5 function but for inclusion of a inhibitor substance. In this fashion, one can measure the
ability of the candidate substance to reduce the tyrosine phosphorylation activity
relatively in the presence of the candidate substance.
"Effective arnounts'~ in certain circllm~n~es are those amounts effective to
3 0 reproducibly reduce neu-tyrosine kinase activity, or to reduce the growth of
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neu-mediated cancer cells, in comparison to their normal levels. Compounds that
achieve significant ~lo~;ate changes in activity will be used. If desired, a battery of
compounds may be screened in vitro to identify second agents for use in the present
invention.
Significant decrease in tyrosine phosphorylation, e.g, as measured using
imml-noblotting techniques with anti-phosphorylation antibodies, are represented by a
reduction in protein phosphorylation levels of at least about 30%-40%, and most
preferably, by decreases of at least about 50%, with higher values of course being
possible. Tyrosine kinase assays that measure tyrosine phosphorylation are well known
in the art and may be conducted in vitro or in vivo.
Inhibition of growth of cancer cells can measured by the MTT assay. A
significant inhibition in growth is represented by decreases of at least about 30%-40% as
co~ ~ed to uninhibited, and most preferably, of at least about 50%, with more
significant decreases also being possible. Growth assays as measured by the MTT assay
are well known in the art. Assays may be conducted as described by Mosmann et al.,
1983; Rubinstein et al., 1990 (incorporated herein by reference). Therefore, if a
candidate substance exhibited inhibition in this type of study, it would likely be a
2 0 suitable compound for use in the present invention.
Qu~~ Live in vitro testing is not a requirement of the invention as it is generally
envisioned that the second agents will often be selected on the basis of their known
~lopt;llies or by structural and/or functional c~-mp~ri~on to those agents disclosed herein.
Therefore, the effective amounts will often be those amounts proposed to be safe for
~fimini~tration to ~nim~ in another context, for example, as disclosed herein. As the
invention arises in part from the inventors' discovery of certain metabolic and
physiological events, and the inventors' surprising combination of elements, there is
considerable information available on the use and doses of second agents alone, which
3 0 information may now be employed with the present invention.
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It will, of course, be understood that all the screening methods of the present
invention are useful in themselves notwithstanding the fact that effective candidates may
not be found. The invention provides methods for screening for such candidates, not
solely methods of finding them.
In still fi~rther embodiments, the present invention is concerned with a method of
inhibiting neu-tyrosine kinase activity which includes subjecting the enzyme to an
effective concentration of an emodin-like inhibitor such as one of the family ofcompounds discussed above, or with a c~n~ t~ substance identified in accordance with
the c~n~licl~te screening assay embodiments. This is, of course, an important aspect of
the invention in that it is believed that by inhibiting the neu tyrosine kinase activity, one
will be enabled to treat various aspects neu-me~ t~(l cancers caused by over-expression
of neu. The use of such emodin-like inhibitors to block neu-tyrosine kinase activity will
serve to treat neu-mediated cancers. The inventors have found that neu-me~ t~rl cancer
cells, which are typically chemoresistant may be sensitized to chemotherapeutic agents.
As such emodin-like tyrosine kinase inhibitors are useful in conjunction with other
chemotherapeutic therapies.
B. Chemother~rellfi~ Agents
A wide variety of chemotherapeutic agents may be used in combination with the
emodin or emodin-like tyrosine kinase inhibitors of the present invention. These can be,
for exarnple, agents that directly cross-link DNA, agents that intercalate into DNA, and
2 5 agents that lead to chromosomal and mitotic aberrations by affecting nucleic acid
synthesis.
Agents that directly cross-link nucleic acids, specifically DNA, are envisaged and
are shown herein, to eventuate DNA damage leading to a synergistic antineoplastic
combination. Agents such as ci~pl~tin, and other DNA alkylating agents may be used.
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Agents that damage DNA also include compounds that hllelre~ with DNA
~, replication, mitosis, and chromosomal segregation. Examples of these compounds
include adriamycin (also known as doxorubicin~, VP-16 (also known as etoposide),v~ ~nil, podophyllotoxin, and the like. Widely used in clinical setting for the
tre~tmtont of neoplasms these compounds are ~f1mini.stered through bolus injections
intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals for adriamycin, to
35-100 mg/m2 for etoposide intravenously or orally
1 0 Ai,lilliotics
Doxorubicin
Doxorubicin hydrochloride, S, 12-N~phthz3renedione~ (8s-cis)- 1 0-[(3-amino-2,3,6-
trideoxy-a-L-lyxo-hexopyranosyl)oxy]-7,8,9, 1 0-tetrahydro-6,8, 1 1 -trihydroxy-8-
(hydroxyacetyl)-1-methoxy-hydrochloride (hydroxydaunorubicin hydrochloride,
Adriarnycin) is used in a wide antineoplastic spectrum. It binds to DNA and inhibits
nucleic acid synthf?si~, inhibits mitosis and promotes chromosomal aberrations.
A~lmini~t.ored alone, it is the drug of first choice for the tre~tment of thyroid
adenoma and primary hepatocellular carcinoma. It is a component of 31 first-choice
combinations for the tre~tment of ovarian, endometrial and breast tumors, bronchogenic
oat-cell carcinoma, non-small cell lung carcinoma, gaskic adenocarcino ma,
retinoblastoma, neuroblastoma, mycosis fungoides, pancreatic carcinoma, prostatic
carcinoma, bladder carcinoma, myeloma, diffuse histiocytic lymphoma, Wilms' tumor,
Hod~kin's (1i~e~e, adrenal tumors, osteogenic sarcoma soft tissue sarcoma, Ewing's
sarcoma, rhabdomyosarcoma and acute lymphocytic lellkemi~ It is an alternative drug
for the trP~tment of islet cell, cervical, testicular and adrenocortical cancers. It is also an
immunosuppressant.
Doxorubicin is absorbed poorly and typically is ~lrnini~tered intravenously. Thepharmacokinetics are multicompartment~l Distribution phases have half-lives of 12
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minllte~ and 3.3 hr. The elimin~tion half~ e is about 30 hr. Forty to 50% is secreted
into the bile. Most of the rem~in~1er is metabolized in the liver, partly to an active
metabolite (doxorubicinol), but a few percent is excreted into the urine. In the presence
of liver imr~irment, the dose should be reduced.
Appropriate doses are, intravenous, adult, 60 to 75 mg/m2 at 21-day intervals or25 to 30 mg/m2 on each of 2 or 3 successive days repeated at 3- or 4-wk intervals or 20
mg/m2 once a week. The lowest dose should be used in elderly patients, when there is
prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow
1 0 invasion, or when the drug is combined with other myelopoietic suppressant drugs. The
dose should be reduced by 50% if the serum bilirubin lies between 1.2 and 3 mg/dL and
by 75% if above 3 mg/dL. The lifetime total dose should not exceed 550 mg/m2 in
patients with normal heart function and 400 mg/m2 in persons havin~ received
me~ tin~l irradiation. Alternatively, 30 mg/m2 on each of 3 consecutive days, repeated
every 4 wk. Exemplary doses may be 10 mg/m2, 20 mg/m2, 30 mg/m2, 50 mg/m2, 100
mg/m2, 150 mg/m2, 175 mg/m2, 200 mg/m2, 225 mg/m2, 250 mg/m2, 275 mg/m2, 300
mg/m2, 350 mg/m2, 400 mg/m2, 425 mg/m2, 450 mg/m2, 475 mg/m2, 500 mg/m2. Of
course, all of these dosages are exemplary, and any dosage in-between these points is
also expected to be of use in the invention.
In the present invention the inventors have employed emodin as an exemplary
emodin-like inhibitor to synergistically enhance the antineoplastic effects of the
doxorubicin in the tre~tmPnt of cancers. Those of skill in the art will be able to use the
invention as exemplified potentiate the effects of doxorubicin in a range of different
neu-me~ t~Dd c~n~rs
Daunorubicin
Daunorubicin hydrochloride, 5,12-Naphthacenedione, (8S-cis)-8-acetyl-10-
[(3-arnino-2~3,6-trideoxy-a-L-lyxo-hexanopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11 -
3 0 trihydroxy- 10-methoxy-, hydrochloride; also termed cerubidine and available from
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Wyeth. Daunorubicin intercalates into DNA, blocks DAN-directed RNA polymerase
and inhibits DNA synthesis. It can prevent cell division in doses that do not interfere
with nucleic acid synthesis.
In combination with other drugs it is included in the first-choice chemotherapy of
acute myelocytic lenkemi~ in adults (for induction of remission), acute lymphocytic
leukemia and the acute phase of chronic myelocytic le--k~mi~ Oral absoIption is poor,
and it must be given intravenously. The half-life of distribution is 45 min~te~ and of
elimin~fion, about 19 hr. The half-life of its active metabolite, daunorubicinol, is about
27 hr. Daunorubicin is metabolized mostly in the liver and also secreted into the bile (ca
40%). Dosage must be reduced in liver or renal insufficiencies.
Suit~ble doses are (base equivalent), intravenous adult, younger than 60 yr. 45
mg/m2/day (30 mglm2 for patients older than 60 yr.) for 1, 2 or 3 days every 3 or 4 wk or
0.8 mg/kg/day for 3 to 6 days every 3 or 4 wk; no more than 550 mg/m2 should be given
in a lifetime, except only 450 mg/m2 if there has been chest irradiation; children, 25
mg/m2 once a week unless the age is less than 2 yr. or the body surface less than 0.5 m,
in which case the weight-based adult srhç~lnle is used. It is available in injectable dosage
forrns (base equivalent) 20 mg (as the base equivalent to 21.4 mg of the hydrochloride).
Exemplary doses may be 10 mg/m2, 20 mg/m2, 30 mg/m2, 50 mg/m2, 100 mg/m2, 150
mg/m2, 175 mg/m~, 2û0 mg/m2, 225 mg/m2, 250 mg/m2, 275 mg/m2, 300 mg/m2, 350
mg/m2, 400 mg/m2, 425 mg/m2, 450 mg/m2, 475 mg/m2, 500 mg/m2. Of course, all of
these dosages are exemplary, and any dosage in-between these points is also expected to
be of use in the invention.
Mitomycin
Mitomycin (also known as mutamycin and/or mitomycin-C) is an antibiotic
isolated from the broth of Streptomyces caespitosus which has been shown to have~ntitllmor activity. The compound is heat stable, has a high meltin~ point, and is freely
3 0 soluble in organic solvents.
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Mitomycin selectively inhi~oits the synthesis of deoxyribonucleic acid (DNA).
The guanine and cytosine content correlates with the degree of mitomycin-inducedcross-linking At high concentrations of the drug, cellular ~NA and protein synthesis are
also suppressed.
In hllm~n~7 mitomycin is rapidly cleared from the serurn after intravenous
~lmini~tration Time re~uired to reduce the serum concentration by 50% after a 30 mg.
bolus injection is 17 minlltes. After injection of 30 mg., 20 mg., or 10 mg. I.~., the
maximal semm concentrations were 2.4 mg./mL, 1.7 mg./mL, and 0.52 mg./mL,
respectively. Clearance is effected primarily by metabolism in the liver, but metabolism
occurs in other tissues as well. The rate of clearance is inversely proportional to the
maximal serum concentration because, it is thought, of saturation of the degradative
pathways.
Approximately 10% of a dose of mitomycin is excreted rlnr.h~nged in the urine.
Since metabolic pathways are saturated at relatively low doses, the percent of a dose
excreted in urine increases with increasing dose. In children, excretion of intravenously
~lmini~tered mitomycin is sirnilar.
Actinomycin D
Actinomycin D (Dactinomycin) ~50-76-0]; C62H86Nl2O~6 (1255.43) is an
antineoplastic drug that inhibits DNA-dependent RNA polymerase. It is a component of
first-choice combinations for trezltm~nt of choriocarcinoma, embryonal
2 5 rhabdomyosarcoma, testicular tumor and Wilms' tumor. Tumors which fail to respond to
systemic treatrnent sometimes respond to local perfusion. Dactinomycin pote~ti~teS
radiotherapy. It is a secondary (efferent) immllnosuppressive.
Actinomycin D is used in combination with primary surgery, radiotherapy, and
3 0 other drugs, particularly vincristine and cyclophosphamide. Antineoplastic activity has
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also been noted in Ewing's tumor, Kaposi's sarcoma, and soft-tissue sarcomas.
Dactinomycin can be effective in women with advanced cases of choriocarcinoma. It
also produces consistent responses in combination with chlorambucil and methotrexate
in patients with metastatic testicular carcin~ m~c A response may snm~times be
~ 5 observed in patients with Hodgkin's disease and non-Hodgkin's Iymphomas.Dactinomycin has also been used to inhibit immunological responses, particularly the
rejection of renal transplants.
Half of the dose is excreted intact into the bile and 10% into the urine; the
hal~-life is about 36 hr. The drug does not pass the blood-brain barrier. Actinomycin D
is supplied as a lyophili7~ powder (0/5 mg in each vial). The usual daily dose is 10 to
15 mg/kg, this is given intravenously for 5 days; if no manifestations of toxicity are
encountered, additional courses may be given at intervals of 3 to 4 weeks. Dailyinjections of 100 to 400 mg have been given to children for 10 to 14 days; in other
re~imen~, 3 to 6 mg/kg, for a total of 125 mg/kg, and weekly mzlin~n~nce doses of 7.5
mg/kg have been used. Although it is safer to ~rlrnini~ter the drug into the tubing of an
intravenous infusion, direct intravenous injections have been given, with the precaution
of discarding the needle used to withdraw the drug from the vial in order to avoid
subcutaneous reaction. Exemplary doses may be 100 mg/m2, 150 mg/m2, 175 mg/m2,
200 mg/m2, ~25 mg/m2, 250 mg/m2, 275 mg/m2, 300 mg/m2, 350 mg/m2, 4()0 mg/m2,
425 mg/m2, 450 mg/m2, 475 mg/m2, 500 mg/m2. Of course, all of these dosages are
exemplary, and any dosage in-between these points is also expected to be of use in the
invention.
2 5 Bleor~ r~
Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolated from a
strain of Streptomyces verticillus. It is freely soluble in water.
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Although the exact mech~ni~m of action of bleomycin is unknown, available
evidence would seem to indicate that the main mode of action is the inhibition of DNA
synthesis with some evidence of lesser inhibition of RNA and protein synthesis.
In mice, high concentrations of bleomycin are found in the sl~in, lungs, kidneys,
peritoneum, and Iymphatics. Tumor cells of the skin and lungs have been found to have
high concentrations of bleomycin in contrast to the low concentrations found in
hematopoietic tissue. The low concentrations of bleomycin found in bone marrow may
be related to high levels of bleomycin degradative enzymes found in that tissue.
In patients with a cre~tinin~ clearance of >35 mL per minute, the serum or plasma
terrnin~l elimin~tion half-life of bleomycin is approximately 115 minlltes In patients
with a cre~tinine clearance of <35 mL per minute, the plasma or serum terrnin~l
elimin~tinn half-life increases exponentially as the creatinine clearance decreases. In
hl~m~n~, 60% to 70% of an ~r1mini~tered dose is recovered in the urine as activebleomycin.
Bleomycin should be considered a palliative treatment. It has been shown to be
useful in the management of the following neoplasms either as a single agent or in
proven combinations with other approved chemotherapeutic agents in squamous cellcarcinoma such as head and neck (including mouth, tongue, tonsil, nasopharynx,
oropharynx, sinus, palate, lip, buccal mucosa, gingiva, epiglottis, larynx), skin, penis,
cervix, and vulva. It has also been used in the ll. c.l,l.cnt of lymphomas and testicular
carcinoma.
Because of the possibility of an ~~ ylactoid reaction, lymphoma patients
should be treated with two units or less for the first two doses. If no acute reaction
occurs, then the regular dosage schedule may be followed.
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Improvement of Hodgkin's Disease and testicular tumors is prompt and noted
within 2 weeks. If no improvement is seen by this time, improvemer~t is unlikely.
Squarnous cell cancers respond more slowly, sometimes re~uiring as long as 3 weeks
before any improvement is noted.
Bleomycin may be given by the intrarnuscular, intravenous, or subcutaneous
routes.
c~ n~ous Agents
Cisplatin
Cisplatin has been widely used to treat cancers such as metastatic testicular orovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung
cancer or other turnors. Cisplatin can be used alone or in combination with other agents,
with efficacious doses used in clinical applications of 15-20 mg/m2 for 5 days every three
weeks for a total of three courses. Exemplary doses may be 0.50 mg/m2, 1.0mg/m2, I.S0
mg/m2, 1.75 mg/m2, 2.0 mg/m2, 3.0 mg/m2, 4.0 mg/m2, 5.0 mg/m2, lOmg/m2. Of
course, all of these dosages are exemplary, and any dosage in-between these points is
also expected to be of use in the invention.
Cisplatin is not absorbed orally and must therefore be delivered via injection
intravenously, subcutaneously, inlldLulllorally or intraperitoneally.
In certain aspects of the current invention cisplatin is used in combination with
emodin or emodin-like compounds in the tre~tment of non-small cell lung carcinoma. It
is clear, however, that the combination of ci~pl~tin and emodin and or emodin-liike
compounds could be used for the trç~tm~nt of any other neu-merli:~te~1 cancer.
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VP16
VP16 is also know as etoposide and is used primarily for treatment of testiculartumors, in combination with bleomycin and cisplatin, and in combination with cisplatin
for small-cell carcinoma of the lung. It is also active against non-Hodgkin's lymphomas,
acute nonlymphocytic lellkemi~ carcinoma of the breast, and Kaposi's sarcoma
associated with acc~uired immunodeficiency syndrome (A~DS).
VP16 is available as a solution (20 mg/ml) for intravenous ~flmini~tration and as
50-mg, liquid-filled capsules for oral use. For small-cell carcinoma of the lung, the
intravenous dose (in combination therapy) is can be as much as 100 mg/m2 or as little as
2 mg/ m2, routinely 35 mg/m2, daily for 4 days, to 50 mg/m2, daily for 5 days have also
been used. When given orally, the dose should be doubled. Hence the doses for small
cell lung carcinoma may be as high as 200-250mg/m2. The intravenous dose for
testicular cancer (in combination therapy) is 50 to 100 mg/m2 daily for 5 days, or 100
mg/m2 on alternate days, for three doses. Cycles of therapy are usually repeated every 3
to 4 weeks. The drug should be ~rlmini~t~red slowly during a 30- to 60-minute infusion
in order to avoid hypotension and bronchospasm, which are probably due to the solvents
used in the formulation.
2 0 Tumor Necrosis Factor
Tumor Necrosis Factor [TNF; Cachectin] is a glyco~lo~ill that kills some kinds
of cancer cells, activates cytokine production, activates macrophages and endothelial
cells, promotes the production of collagen and coll~g~n~ç~, is an infl~mm~tory mediator
and also a mefli~tor of septic shock, and promotes catabolism, fever and sleep. Some
infectious agents cause tumor regression through the stim~ tion of TNF production.
TNF can be quite toxic when used alone in effective doses, so that the optimal regimens
probably will use it in lower doses in combination with other drugs. Its
immlmosuppressive actions are pot~nti~tf ~I by gamma-interferon, so that the combination
potentially is dangerous. A hybrid of TNF and interferon-oc also has been found to
3 0 possess anti-cancer activity.
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Plant Alkaloids
Taxol
Taxol is an experimental antimitotic agent, isolated from the bark of the ash tree,
Taxus brevifolia. It binds to tubulin (at a site distinct from that used by the vinca
alkaloids) and promotes the assembly of microtubules. Taxol is currently being
evaluated clinically; it has activity against m~lign~nt melanoma and carcinoma of the
ovary. ~cims~l doses are 30 mg/m2 per day for 5 days or 210 to 250 mg/m2 given once
everv 3 weeks. Of course, all of these dosages are exemplary, and any dosage in-between
these points is also expected to be of use in the invention.
Vincr~stine
Vincristine blocks mitosis and produces met~rh~ce arrest. It seems likely that
most of the biological activities of this drug can be explained by its ability to bind
specifically to tubulin and to bloclc the ability of protein to polymerize into microtubules.
Through disruption of the microtubules of the mitotic ~:udL~lS, cell division is arrested
in metaphase. The inability to segregate chromosomes correctly during mitosis
presumably leads to cell death.
The relatively low toxicity of vincristine for normal marrow cells and epithelial
cells make this agent unusual arnong anti-neoplastic drugs, and it is often included in
combination with other myelosuppressive agents.
2 5 Unpredictable absorption has been reported after oral ~iministration of
vinblastine or vincristine. At the usual clinical doses the peak concentration of each drug
in plasma is approximately 0.4 mM.
Vinblastine and vincristine bind to plasma proteins. They are extensively
3 0 concentrated in platelets and to a lesser extent in leukocytes and erythrocytes.
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Vincristine has a multiphasic pattern of clearance from the plasma; the terminalhalf-life is about 24 hours. The drug is metabolized in the liver, but no biologically
active derivatives have been identified. Doses should be reduced in patients with hepatic
dysfunction. At least a 50% reduction in dosage is indicated if the concentration of
bilirubin in plasma is greater than 3 mg/dl (about 50 mM).
Vincristine sulfate is available as a solution (1 mg/ml) for intravenous injection.
Vincristine used together with corticosteroids is presently the tre~tm~nt of choice to
induce remissions in childhood leukemia, the optimal dosages for these drugs appear to
be vincristine, intravenously, 2 mg/m2 of body-surface area, weekly, and prednisone,
orally, 40 mg/m~, daily. Adult patients with ~Iodgkin's disease or non-Hodgkin'sIymphomas usually receive vincristine as a part of a complex protocol. When used in the
MOPP regimen, the recommended dose of vincristine is 1.4 mg/m~. High doses of
vincristine seem to be tolerated better by children with lel-k~mi~ than by adults, who may
experience sever neurological toxicity. Administration of the drug more frequently than
every 7 days or at higher doses seems to increase the toxic manifestations without
proportional improvement in the response rate. Precautions should also be used to avoid
extravasation during intravenous ~-lmini~tration of vincristine. Vincristine (and
2 0 vinblastine) can be infused into the arterial blood supply of tumors in doses several times
larger than those that can be ~lmini~t~red intravenously with comparable toxicity.
Vincristine has been effective in Hodgkin's disease and other Iymphomas.
Although it appears to be somewhat less beneficial than vinblastine when used alone in
Hodgkin's rli~e~e, when used with mechlo~ 7 prednisone, and procarbazine (the
so-called MOPP regimen), it is the preferred treatment for the advanced stages (III and
IV) of this disease. In non-Hodgkin's Iymphomas, vincristine is an important agent,
particularly when used with cyclophosphamide, bleomycin, doxorubicin, and prednisone.
Vincristine is more useful than vinblastine in Iymphocytic leukemia. Benef1cial
response have been reported in patients with a variety of other neoplasms, particularly
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Wilms' tumor, neuroblastoma, brain tumors, rhabdomyosarcoma, and carcinomas of the
breast, bladder, and the male and female reproductive systems.
~oses of vincristine for use will be detrrrnin~1 by the clinician according to the
individual patients need. 0.01 to 0.03mg/kg or ().4 to 1.4mg/m2 can be ~1mini~red or
1.5 to 2mg/m2 can also be ~ mini~tered. .Altern~ively 0.02 mg/m2, 0.05 mg/m2, 0.06
mg/m~, 0.07 mg/m2, 0.08 mg/m2, 0.1 mglm2, 0.12 mg/m2, 0 14 mg/m2, 0.15 mg/m2, 0.2
mg/m2, 0.25mg/m2 can be given as a constant intravenous infusion. Of course, all of
these dosages are exemplary, and any dosage in-between these points is also expected to
be of use in the invention.
Vinblastine
When cells are incubated with vinblastine, dissolution of the microtubules occurs.
Unpredictable absoIption has been reported after oral ~-lmini~,tration of vinblastine or
vincristine. At the usual clinical doses the peak concentration of each drug in plasma is
approximately 0.4 mM. Vinblastine and vincristine bind to plasma proteins. They are
sively concentrated in platelets and to a lesser extent in leukocytes and erythrocytes.
After intravenous injection, vinblastine has a multiphasic pattern of clearance
from the plasma; after distribution, drug disappears from plasma with half-lives of
approximately 1 and 20 hours.
Vinblastine is metabolized in the liver to biologically activate derivative
desacetylvinblastine. Approximately 15% of an ~rlmini~tered dose is detected intact in
the urine, and about 10% is recovered in the feces after biliary excretion. Doses should
be reduced in patients with hepatic dysfunction. At least a 50% reduction in dosage is
indicated if the concentration of bilirubin in plasma is greater than 3 mg/dl (about S0
mM).
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Vinblastine sulfate is available in preparations for injection. The drug is given
intravenously; special precautions must be taken against subcutaneous extravasation,
since this may cause painful irritation and ulceration. The drug should not be injected
into an extremity with impaired circulation. After a single dose of 0.3 mg/kg of body
weight, myelosuppression reaches its maxi~llu-ll in 7 to 10 days. lf a moderate level of
leukopenia (approximately 3000 cells/mm3) is not ~tt~in~-1, the weekly dose may be
increased gradually by increments of 0.05 mg/kg of body weight. In regimens df-sign~d
to cure testicular cancer, vinblastine is used in doses of 0.3 mg/kg every 3 weeks
irrespective of blood cell counts or toxicity.
The most important clinical use of vinblastine is with bleomycin and cisplatin in
the curative therapy of metastatic testicular tumors. Beneficial responses have been
reported in various lymphomas, particularly Hodgkin's ~ e~ce~ where significant
improvement may be noted in 50 to 90% of cases. The effectiveness of vinblastine in a
high proportion of lymT)h~-m~s is not ~limini~hed when the disease is refractory to
alkylating agents. lt is also active in Kaposi's sarcoma, neuroblastoma, and
Letterer-Siwe disease (histiocytosis ~), as well as in carcinoma of the breast and
choriocarcinoma in women.
2 0 Doses of vinblastine for use will be determined by the clinician according to the
individual patients need. 0.1 to 0.3mg/kg can be zl~lmini~tered or 1.5 to 2mg/m2 can also
be ~lmini~tered Altern~tively, 0.1 mg/m2, 0.12 mg/m2, 0.14 mg/m2, 0.15 mg/m2, 0.2
mg/m2, 0.25 mgfm2, 0.5 mg/m2, 1.0 mg/m2, 1.2 mg/m2, 1.4 mg/m2, 1.5 mg/m2, 2.0
mg/m2, 2.5 mg/m2, 5.0 mg/m2, 6 mg/m2, 8 mg/m2, 9 mg/m2, 10 mg/m2, 20 mg/m2, can be
2 5 given. Of course, all of these dosages are exemplary, and any dosage in-between these
points is also expected to be of use in the invention.
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Alkylating Agents
Carmusfine
Carmustine (sterile carmustine) is one of the nitrosoureas used in the treatment of
certain neoplastic rli~e~es It is 1,3bis (2-chloroethyl3-l-nitrosourea. It is Iyophilized
pale yellow flakes or congealed mass with a molecular weight of 214.06. It is highly
soluble in alcohol and lipids, and poorly soluble in water. Carrnustine is ~lmini~t~red by
intravenous infusion after reconstitution as recommenc~P!i The structural for~ula is:
fj)
Cl--CH 2--CH 2~ C--NH--CH 2--CH 2--t~l
NO
Sterile c~rmll~tine is commonly available in 100 mg single dose vials of
Iyophilized m~terizll
Although it is generally agreed that carmustine alkylates DNA and RNA, it is notcross resistant with other alkylators. As with other nitrosoureas, it may also inhibit
2 0 several key enzymatic processes by carbamoylation of amino acids in proteins.
C~ stin~ is indicated as palliative therapy as a single agent or in established
combination therapy with other approved chemotherapeutic agents in brain tumors such
as glioblastoma, br~in~fem glioma, medullobladyoma, astrocytoma, ependymoma, andmet~ct~tic brain tumors. Also it has been used in combination with prednisone to treat
multiple myeloma. Carmustine has proved useful, in the tre~tment of Hodgkin's Disease
and in non-Hodgkin's lymphomas, as secondary therapy in combination with other
approved drugs in patients who relapse while being treated with primary therapy, or vvho
fail to respond to primary therapy.
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The recommended dose of carmustine as a single agent in previously untreated
patients is 150 to 200 mg/m2 intravenously every 6 weeks. This may be given as a single
dose or divided into daily injections such as 75 to 100 mg/m2 on 2 successive days.
When carmustine is used in combination with other myelosuppressive drugs or in
patients in whom bone marrow reserve is depleted, the doses should be adjusted
accordingly. Doses subsequent to the initial dose should be adiusted according to the
hematologic response of the patient to the prece~inp; dose. It is of course understood that
other doses may be used in the present invention for example lOmg/m2, 20mg/m2,
30mg/m2 40mg/m2 SOmg/m2 60mg/m~ 70mg/m2 80mg/m2 90mg/m2 lOOmg/m2 . The
skilled artisan is directed to, "Remington's Ph~rrn~c.eutical Sciences" 1 5th Edition,
chapter 61. Some variation in dosage will necessarily occur depending on the condition
of the subject being treated. The person responsible for ~imini~tration will, in any event,
deterrnine the appropriate dose for the individual subject
1 5 Melp~talan
Melphalan also known as alkeran, L-phenyl~l~nine mustard, phenyl~ nine
mustard? L-PAM, or L-sarcolysin, is a phenylalanine derivative of nitrogen mustard.
Melphalan is a bifunctional alkylating agent which is active against selective human
neoplastic diseases. It is known chemically as
2 0 4-[bis(2-chloroethyl)amino]-L-phenylalanine.
Melphalan is the active L-isomer of the compound and was first synthesi~ed in
1953 by Bergel and Stock; the D-isomer, known as medphalan, is less active against
certain animal tumors, and the dose needed to produce effects on chromosomes is larger
2 5 than that required with the L-isomer. The racemic (DL-) form is known as merphalan or
sarcolysin. Melphalan is insoluble in water and has a pKal of~2.1. Melphalan is
available in tablet form for oral a~lrnini~tration and has been used to treat multiple
myeloma.
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Available evidence suggests that about one third to one half of the patients with
multiple myeloma show a favorable response to oral a~lmini~tration of the drug.
Melphalan has been used in the tre~tment of epithelial ovarian carcinoma. One
commonly employed regimen for the treatment of ovarian carcinoma has been to
mini~ter melphalan at a dose of 0.2 mg/kg daily for five days as a single course.
Courses are repeated every four to five weeks depending upon hematologic toleraxlce
(Smith and Rutledge, 1975; Young et al., 1978). Alternatively the dose of melphalan
used could be as low as 0.05mg/kg/day or as high as 3mg/kg/day or any dose in between
these doses or above these doses. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person responsible for
~lmini~tration will, in any event, determine the ~u~liate dose for the individual
subject
1 5 Cyc~op~tosp~tamide
Cyclophosphamide is 2H- 1,3 ,2-Oxazaphosphorin-2-amine,
N,N-bis(2-chloroethyl)tetrahydro-, 2-oxide, monohydrate; termed Cytoxan available
from Mead Johnson; and Neosar available from Adria. Cyclophosphamide is preparedby condensing 3-amino- 1 -propanol with N,N-bis(2-chlorethyl) phosphoramidic
dichloride [(ClCH2CHz)2N--POCl2] in dioxane solution under the catalytic influence of
triethylarnine. The conflen~tion is double, involving both the hydroxyl and the amino
groups, thus effecting the cyclization.
Unlike other 13-chloroethylamino alkylators, it does not cyclize readily to the
active ethyleneimonium form until activated by hepatic enzymes. Thus, the substance is
stable in the gastrointestinal tract, tolerated well and effective by the oral and parental
routes and does not cause local vesication, necrosis, phlebitis or even pain.
Suitable doses for adults include, orally, 1 to 5 mg/kg/day (usually in
3 0 combination), depending upon gastrointestinal tolerance; or 1 to 2 mg/kg/day;
..
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intravenously, initially 40 to 50 mg/kg in divided doses over a period of 2 to 5 days or 10
to 15 mg/kg every 7 to 10 days or 3 to 5 mg/kg twice a week or 1.5 to 3 mg/kg/day . A
dose 250mg/kg/day may be ~rlmini~tered as an antineoplastic. Because of
gastrointestinal adverse effects, the intravenous route is ~l~r~lled for loading. During
m~inter ~nce, a leukocyte count of 30ûO to 4000/mm3 usually is desired. The drug also
sometimes is ~imini~tered intramuscularly, by infiltration or into body cavities. It is
available in dosage forms for in~ection of 100, 200 and 500 mg, and tablets of 25 and 50
mg the skilled artisan is referred to "Remington's Ph~ reutical Sciences" 1 5th Edition,
chapter 61, incorporate herein as a reference, for details on doses for ~-lmini~tration.
C~lloram~ucil
Chlorambucil (also known as leukeran) was first synth~si71--l by Everett et al.
(1953). It is a bifunctional alkylating agent of the nitrogen mustard type that has been
found active against selected human neoplastic diseases. Chlorambucil is kno~,vnchemically as 4-[bis(2-chlorethyl)amino] benzenebutanoic acid.
Chlorambucil is available in tablet form for oral ?111mini~tration. It is rapidly and
completely absorbed from the gastrointestinal tract After single oral doses of 0.6-1.2
mg/kg, peak plasma chlorambucil levels are reached within one hour and the terrnin~l
half-life of the parent drug is ~stim~t~l at 1.5 hours. 0.1 to 0.2mg/kg/day or 3 to
6mglm2/day or alternatively 0.4mg/kg may be used for antineoplastic treatment.
Trç~tment regimes are well lcnow to those of skill in the art and can be found in the
"Physicians Desk Reference" and in "Remingtons Ph~ ce~lticzll Sciences" referenced
herem.
Chlorambucil is indicated in the treatment of chronic Iymphatic (Iymphocytic)
leukemia, m~lign~nt lymphomas including Iymphosarcoma, giant follicular lymphomaand Hodgkin's disease. It is not curative in any of these disorders but may produce
clinically useful palliation.
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Busulfan
Busulfan (also known as myleran) is a bifunctional alkylating agent. Busulfan isknown chemically as 1,4-butanediol dimethanesulfonate.
Busulfan is not a structural analog of the nitrogen mustards. Busulfan is available
in tablet form for oral ~imini~tration. Each scored tablet contains 2 mg busulfan and the
inactive ingredients m~gn~.sjum stearate and sodium chloride.
Busulfan is indicated for the palliative treatment of chronic myelogenous
~myeloid, myelocytic, granulocytic) lellkemi~ Although not curative, busulfan red~lces
the total granulocyte mass, relieves symptoms of the disease, and improves the clinical
state of the patient. Approximately 90% of adults with previously untreated chronic
myelogenous ~eukemia will obtain hematologic remission with regression or stabilization
of organomegaly following the use of busulfan. It has been shown to be superior to
splenic irradiation with respect to survival times and m~in~en~nce of ~emoglobin levels,
and to be equivalent to irradiation at controlling splenomegaly.
Lomustine
Lomustine is one of the nitrosoureas used in the treatment of certain neoplasticdiseases. It is 1-(2-chloro-ethyl)-3-cyclohexyl-1 nitrosourea. It is a yellow powder with
the empirical forrnula of CgHl6ClN3O2 and a molecular weight of 233.71. Loml~tine is
soluble in 10% ethanol (O.OS mg per mL) and in absolute alcohol (70 mg per mL).
Lomll~tine is relatively insoluble in water (<O.OS mg per mL). It is relatively unionized
at a physiological pH. Inactive ingredients in loml-~tine capsules are: m~gnPsiurn
2 5 stearate and mzlnnitol.
Although it is generally agreed that lomllctine alkylates DNA and RNA, it is notcross resistant with other alkylators. As with other nitrosoureas, it may also inhibit
,~ several key enzymatic processes by carbamoylation of amino acids in proteins.
,,
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Lomustine may be given orally. ~ollowing oral ~llmini~tration of radioactive
lomll~tine at doses ranging from 30 mg/m2 to 100 mg/m2, about half of the radioactivity
given was excreted in the form of degradation products within 24 hours.
The serum half-life of the metabolites ranges from 16 hours to 2 days. Tissue
levels are comparable to plasma levels at 15 mimlte~ after intravenous ~flmini.~tration.
Lomustine has heen shown to be useful as a single agent in addition to other
treatment modalities, or in established combination therapy with other approved
chemothe.~eulic agents in both primary and metastatic brain tumors? in patients who
have already received a~ pl;ate surgical and/or radiotherapeutic procedures. It has also
proved effective in secondary therapy against Hodgkin's Disease in combination with
other approved drugs in patients who relapse while being treated with primary therapy, or
who fail to respond to primary therapy.
The recommended dose of lomllstine in adults and children as a single agent in
previously untreated patients is 130 mg/m2 as a single oral dose every 6 weel~s. In
individuals with co~ lllised bone marrow function, the dose should be reduced to 100
mg/m2 every 6 weeks. When lomustine is used in combination with other
2 0 myelosuppressive drugs, the doses should be adjusted accordingly. It is understood that
other doses may be used for example, 20mg/m2 30mg/m2, 40 mg/m2, SOmg/m2,
60mg/m2, 70mg/m2, 80mg/m2, 90mg/m2, lOOmg/m2, 120mg/m2 or any doses between
these figures as determined by the clinician to be necess~ry for the individual being
treated.
C. ~ ti~l Compositions and Routes of ~.~ al;on
Aqueous compositions of the present invention will have an effective amount of
emodin or emodin-like compound alone or in combination with an effective amount of a
compound (sccond agent) that is a chemotherapeutic agent as exemplified above. Such
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compositions will generally be dissolved or dispersed in a ph~ ceutically acceptable
carrier or aqueous medium.
The phrases "ph~ celltically or ph~rm~ ologically acceptable" refer to
molecular entities and compositions that do not produce an adverse, allergic or other
untoward reaction when ~imini~tered to an animal, or human, as a~ iate. As used
herein, "ph~ eutically acceptable carrier" includes any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying
agents and the like. The use of such media and agents for ph~rm~(~eutical activesubstances is well known in the art. ~xcept insofar as any conventional media or agent is
incompatible with the active ingredients, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients, such as other anti-cancer agents, can
also be incorporated into the compositions.
In addition to the compounds fonn~ t~l for parenteral Z~ imini.~tration, such asintravenous or intramuscular injection, other ph~rm~çeutically acceptable forms include,
e.g., tablets or other solids for oral ~iministration; time release capsules; and any other
form currently used, including cremes, lotions, mouthwashes, inhalents and the like.
2 0 P~ ini~tration
The active compounds of the present invention will often be formulated for
parenteral a~imini~tration, e.g, forrnulated for injection via the intravenous,
intrtlmu~c~ r, sub-cutaneous, or even intraperitoneal routes. The preparation of an
2 5 aqueous composition that contains emodin or emodin-like compounds alone o r in
combination with a second agent as active ingredients will be known to those of skill in
the art in light of the present disclosure. Typically, such compositions can be prepared as
injectables, either as liquid solutions or suspensions; solid forms suitable for using to
prepare solutions or suspensions upon the addition of a liquid prior to injection can also
3 0 be prepared; and the p~ ions can also be emulsified.
J
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Solutions of the active compounds as free base or ph~rm~ologically acceptab1e
salts can be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions can also be pre~a,~d in glycerol, ~iquid
polyethylene glycols, and mixtures thereof and in oils. I~nder ordinary conditions of
storage and use, these ~rep~dlions contain a preservative to prevent the growth of
microorg~ni ~m ~
The ph~rrn~çeutical forrns suitable for injectable use include sterile aqueous
solutions or dispersions; formulations including sesame oil, peanut oil or aqueous
propylene glycol; and sterile powders for the extemporaneous ~ tion of sterile
injectable solutions or dispersions. In all cases the form must be sterile and must be fluid
to the extent that easy syringability exists. It must be stable under the conditions of
m~n11f~cture and storage and must be preserved against the cont~min~1ing action of
microor~ni~m~, such as bacteria and fungi.
The active compounds may be forrnulated into a composition in a neutral or salt
forrn. Pharrnaceutically acceptable salts, include the acid addition salts (formed with the
free amino groups of the protein) and which are forrned with inorganic acids such as, for
exarnple, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic,
2 0 tartaric, mandelic, and the like. Salts forrned with the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium, pol~1csi1.,", ammoniurn,
calciurn, or ferric hydroxides, and such organic bases as isopropylarnine, trimethylamine,
histidine, procaine and the like.
The carrier can also be a solvent or dispersion medium cont~ining, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity
can be n~int~ined, for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion and by the use of
surf~ct~nt.~. The prevention o~ the action of microor~ni.cm~ can be brought about by
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various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phen,ol,
sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include
- isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the compositions of agents
" 5 delaying absorption, for example, alurninum monostearate and gelatin.
Sterile in3ectable solutions are prepared by incorporating the active compounds in
the required arnount in the a~propliate solvent with various of the other ingredients
enumerated above, as required, followed by filtered sterilization. Generally, dispersions
are prepared by incorporating the various sterilized active ingredients into a sterile
vehicle which contains the basic dispersion medium and the required other ingredients
from those enumerated above. In the case of sterile powders for the ~ aldlion of sterile
injectable solutions, the preferred methods of ~lepaldLion are vacuurn-drying and
freeze-drying techniques which yield a powder of the active ingredient plus any
additional desired ingredient from a previously sterile-filtered solution thereof.
In certain cases, the therapeutic formulations of the invention could also be
prepared in forms suitable for topical ~lmini~tration, such as in cremes and lotions.
These forms may be used for treating skin-associated ~ e~ces~ such as various sarcomas.
Upon formulation, solutions will be ~rlministered in a manner compatible with
the dosage formulation and in such arnount as is therapeutically effective. 7rheformulations are easily ~lmini~t~red in a variety of dosage forms, such as the type of
inJectable solutions described above, with even drug release capsules and the like being
2 5 employable.
For parenteral ~lministration in an aqueous solution, for example, the solution
should be suitably buffered if nec~ss~ry and the liquid diluent first rendered isotonic with
sufficient saline or glucose. These particular aqueous solutions are especially suitable for
3 o intravenous, intramuscular, subcutaneous and intraperitoneal ~-7ministration. In this
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connection, sterile aqueous media which can be employed will be known to those of skill
in the art in light of the present disclosure. ~or example, one dosage could be dissolved
in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid
or injected at the proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some
variation in dosage will necessarily occur depending on the condition of the subject being
treated. The person responsible for ~tlmini~tration will, in any event, determine the
~lol.liate dose for the individual subject.
D. Kits
All the essential m~teri~ and reagents required for inhibiting tumor cell
proliferation may be assembled together in a kit. When the components of the kit arc
provided in one or more liquid solutions, the liquid solution preferably is an aqueous
solution, with a sterile aqueous solution being particularly preferred.
For in vivo use, emodin or emodin-like compound, alone or in combination with,
a chemotherapeutic agent may be form~ t.o~ into a single or separate phz~ el~tically
acceptable syringeable composition. In this case, the container means may itself be an
inh~ nt, syringe, pipette, eye ~ pe., or other such like a~pa~dL~Is, from which the
formulation may be applied to an infected area of the body, such as the lungs, injected
into an animal, or even applied to and mixed with the other components of the kit.
The components of the kit may also be provided in dried or lyophili7Pd forms.
2 5 When reagents or co~ Jonents 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 container means. The kits of the invention may also include an instruction
sheet defining ~lmini~tration of the emodin-like tyrosine kinase inhibitor and/or the
chemotherapeutic drug.
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The kits of the present invention also will typically include a means for
cont~inin~ 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 containers, the kits of the invention also may comprise, or be
- 5 packaged with, an instrument for assisting with the injection/~mini~tration or placement
of the ultimate complex coml)osiLion within the body of an animal. Such an instrument
may be an inh~l~n1, syringe, pipette, forceps, measured spoon, eye dropper or any such
medically approved delivery vehicle.
~ ~ ~
EXAMPLE 1
Methodology
1 5 This example relates to the methods used in the current invention. Those of skill
in the art will be able to adapt these methods to the specific requirements of each system
Cell lines and culture. Human breast cancer cell lines MDA-MB453, BT-483,
MDA-MB231, MCF-7 and immortalized breast cell line HBL-100 were obtained from
the American Type Culture Collection (Rockville, MD). AU-565 cells were obtainedfrom the Naval Bioscience Laboratory (Oakland, CA). MDA-MB453, BT483 and
AU-565 cells ove~ ess neu whereas MDA-MB231, MCF-7, and HBL-100 cells
express basal level of neu.
Human lung cancer cell lines NCI-H226, NCI-H1435, and NCI-H460 were also
obtained from the American Type Culture Collection (Rockville, MD). NCI-H1435 cells
o~re~l e;,~ neu, whereas NCI-H226 and NCI-H460 cells express very low levels of neu
(Tsai et al., 1993). H460.eB2, H460.eB3 and H460.neo are stable transfectants derived
from NCI-460 cells (Yu et al., 1994). H460.eB2 and H460.eB3 express high levels of
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neu expressing vector. H460.neo contains only pSV2-neo drug-selection plasmid and
serves in this study as a control (Yu et a~., 1994).
All cells were grown in Dulbeccos's modified Eagle's medium/F12 (C;IBCO,
Grand island, NY) supplemented with 10% fetal bovine serum and gentamicin (50
mg/ml). Cells were grown in a humidified incubator at 37~C under 5% CO2 in air.
Immunoblotting. As described previously (Yu et al., 1990), cells treated
overnight in the presence or absence of emodin and in the absence of serum were washed
three times with PBS and then }ysed in Iysis buffer (20 mM Na2 PO4, pH 7.4; 150 mM
NaCl, 1% Triton X-100; 1% a~ Li~ ; 1 mM phenylmethysulfonyl fluoride; 10 mg/ml
leupeptin; 100 mM NaF and 2 mM Na3VO4). Protein contents were deterrnined against a
standardized control using the Bio-Rad protein assay kit (Bio-Rad Laboratories,
Hercules, CA). A totai of 500 mg of protein was separated by 6% SDS-PAGE and
transferred to nitrocellulose filter paper (Schleicher & Schuell, Inc., Keene, NH).
Nonspecific binding on the nitrocellulose filter paper was minimi7~-1 with a blocking
buffer c~ non-fat dry milk (5%) and Tween ~0 (0.1%, v/v) in P13S (PBS/Tween
20). The treated filter paper was inc~lb~te(l with primary antibodies (the anti-pl85neU
antibody c-neu [Ab-3] for detection of pl85neU or the anti-phosphotyrosine antibody
2 0 [UBI, Lalce Placid, NY] for detection of phosphotyrosine), and incubated with HRP-goat
anti-mouse antibody (1:1000 dilution) (Boehringer ~nnheim Corp., Tncli~n~polis, IN).
Bands were vi~n~ l with the enhanced Chemil~lmin~scence system (Amersham Corp.,
Arlington Heights, IL~.
2 5 Immuno-complex Kinase Assay. The immlln~-complex kinase assay was
modified from those described previously (Kiyokawa et al., 1995). Briefly, cells were
treated with or without 40 !1~ emodin for 24 h, then washed 3 times with PBS. Cells
were then collected and lysed in lysis buffer. Cell lysates (500 mg) were incubated with
monoclonal anti-p185neU antibody c-neu ~Ab-3) for 1 h at 4~C, then precipitated with 50
3 0 ml of protein-A-conjugated agarose (Boehringer ~nnheim) for 30 minutes at 4~C, and
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washed 3 times with 50 mM Tris-HCl buffer cont~ining 0.5 M LiCl ~pH 7.5) and once in
assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MnCl2). To 40 ml of beads
- (protein-a-conjugated agrose), 10 ~Ci of [7~-32P]ATP (Amersham) and 10 ml of enolase
(Sigma Chemical Co., St. Louis, MO) were added and in~ b~t~-1 for 20 minutes at room
temperature. The re~ct~nt~ were separated by 7.5% SDS-PAGE. The gel was dried and
vi~ li7e~1 by autoradiography.
Proliferation assay. Cells were detached by trypsinization, seeded at 2 x 104
cells/ml in a 96-well microtiter plate overnight, then treated with various concentrations
of test samples and incubated for an additional 72 h. The effects on cell growth of
emodin~ cisplatin, doxorubicin, or VP16 alone or in combination were f Y~mined by Ml~
assay (Mosmann et al., 1983; Rubinstein et al., 199Q). Briefly, 20 ~Ll of MTT solution (5
mg/ml) (Sigma Chemical Co., St. Klouism, MO) was added to each well and incubated
for 4 h at 37~C. The supernatant was aspirated, and the MTT formazan formed by
metabolical viable cells was dissolved in 150 ~11 of dimethyl sulfoxide, then monitored
by a microplate reader (Dynatech MR 5000 fluorescçnce, Dynatech Corp., Burlinglton,
MA) at a wavelength of 590 nm.
Colony Formation in Soft Agarose. As described previously (Yu et al., 1993),
cells (1 x 103 cells/ml3 were seeded in 24-well plates in culture medium Cont~ining
0.35% agarose (FMC Corp., Rockland, ME) over a 0.7% agarose layer and incubated for
3 weeks at 37~C. Colonies were then stained with p-iodonitrotetrazolium violet
(lmg/ml), and colonies larger than 100mM were counted. Each rlet~rrnin~tion was
performed 4 times.
Lipid Vis ~li7~tion. As described previously (Sheehan, 1980) a modified Oil
Red O in propylene glycol method was used to visualize neutral lipids (Bacus et al.,
1992; Bacus et al., 1990).
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Evaluation of drug combination. For evaluating the combined effect of the two
drugs, observed values were compared to predicted values (c) calculated from theequation c = a x b/100, where a and b indicate survival values with single agents (Webb,
1963; Hata et a/., 1994). Observed values of less than 70% of predicted ones were
considered synergistic.
Zymography of Gelatinolytic Activity. As described previously (Yu and Hung,
1991). Cells were ~et~ch~cl by trypsinization, seeded at 2 x 106 cells/well in a 6-well
plate and cultured in DMEM/F12 medium supplemented with 1% FBS for overnight,
washed cells with PBS, added serum free DMEM/F12 medium, then treated with various
concentrations of test samples and incubated for an additional 24 h. The culturesupernatants were collected, centrifuged at 800 x g for 10 min, then at 18,000 x g for 10
min. The supernatants ( 150 ~11) were analyzed by zymography using SDS-PAGE
contSlining 1.5% gelatin prepared according to procedures described previously (Yu and
Hung, 19913.
In Vitro Chemoinvasion Assay. In vitro invasiveness was carried out according
to the procedure described previously (Yusa et al., 1990; 1994) with modifications,
Briefly, 24 well Transwell unites with an 8-~m pore size polycarbonate filter (Costar
Corp., Carnbridge, MA) were coated with 0.1 ml of a 1:30 dilution (48 ~lg/filter) of
Matrigel (Yusa et al., 1990; Yu et al., 1994) in cold DMEM/F 12 medium, then air dried
these filters at room temperature, and forrned a thin continuous layer on top of the filter.
The lower co~ ent contained 0.6 ml l~minin (20 mg/ml, Becton Dickinson) as
chemoattractant or DMEM/F12 medium as a negative control. The cells (1 x 105
cells/0.1 ml of DMEM/F12 co"l~i";~ 0.1% bovine serum albumin) were placed in theupper co,l,~a~Ln,ent and incubated with or without either emodin or DK-V-47 for 72 h at
37 ~C in a humidified 95% air, 5% C02. After the incubation, the filters were fixed with
3% glutaraldehyde in PBS and stained with Giemsa, then counted the nurnber of cells per
High-power field (X 200) that had migrated to the lower side of the filter.
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EXAMPLE 2
Effect of Emodin on Tyrosine Phosphorylation in
Breast Cancer Cells that Overexpress neu
To test whether emodin, a tyrosine kinase inhibitor for the protein tyrosine kinase
p56/ck (Jayasuriya et al., 1992), may also inhibit neu tyrosine kinase, human breast cancer
cells MDA-MB453 that OVt;lc~ S pl85neU were used to test the effect of emodin ontyrosine phosphorylation of pl85neU.
Cells were treated with different concentrations of emodin at 37~C for 24 h, then
analyzed for the protein level of pl85neU and its tyrosine phosphorylation. The pl85neU
was first immunoprecipitated by anti-pl85neU antibody and the imml~noprecipitates were
then blotted with anti-phosphotyrosine antibody for detection of phosphotyrosine or
anti-pl 85neU antibody for pl 85neU detection.
Emodin, at a 40 ~M concentration, in~ cetl a ~i~nific~nt reduction in the l~vel of
tyrosine phosphorylation (FIG. lA), but had no effect on pl85neU protein level. The
reduced tyrosine phosphorylation of pl85neU could be readily detected after 12 h (FIG.
lB).
To further confirrn that the reduced tyrosine phosphorylation by emodin is a
general phenomenon for pl85neU, other neu-overex~lt;s~ g breast cancer cell lines were
also ~ min~cl and similar results were obtained (FIG. 2). These cell lines include
BT-483, AU-565 (FIG. 2), SKBr-3, and MDA-MB361.
When MCF-7 cells that express basal level of pl85neU protein were ex~min~cl
similarly, phosphotyrosine level of pl85neU was almost undetectable under the
experimental condition. This makes it in~ignificant to compare the effect of emodin on
phosphotyrosine level on pl 85neU in MCF-7 cells.
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To test relationship between chemical structure and inhibitory activity of emodin
and its derivEives on tyrosine phosphorylation of HER-2/neu and proliferation ofHER-2/nell-ov~,ie~ ssing breast cancer cells, 12 derivatives of emodin as shown in
Table 1, were synthesized and according to substituent at the different structure position
of emodin, these derivatives were separated into five groups. Human breast cancer
MDA-MB 453 cells which ovelexL~less pl85neU were treated with varying
concentrations of emodin and 12 derivatives at 37~C ~or 24 hr, then analyzed for the
protein level of pl85neU and its tyrosine phosphorylation using immllnoblotting with
anti-phosphotyrosine antibody for detection of phosphotyrosine (anti-PY) or
anti-pl85neU antibody for detection of p185neU.
As show in the Table 2, in all compounds tested, emodin's carbon 9 (C9) group
substitute, named DK-V-47 is the most effective to suppress tyrosine phosphorylation of
p 1 85neU, the parental compound emodin is less effective than DK-V-47. The
concentration of emodin and DK-V-47 needed for 50% inhibitory activity of tyrosine
phosphorylation are 21 ~M, and 17 ,uM, respectively. Under the sarne condition, emodin
and its derivatives did not affect protein levels of p 1 85nell. A nurnber of other inhibitors
listed herein below also had a marked inhibitory on prolifior~tion of cells even though
their
Table 2. Relationship between chemical structure of emodin and its
de~ lives and inhibitory activity Oll tyrosine phosphorylation of neu and
proliferation of MDA-MB 453 cells. Cells in the serum-free medium were incubatedwith emodin or it's derivatives (0, 10, 20, 30, 40, 60, 80 and 100 ,uM) at 37~C for 24 h,
2 5 then blotted with antiphosphotyrosine antibody and quantified with density scanner
program, (b), cells were treated with different concentrations of emodin and derivatives
for 72 h. The effect on cell growth was e~mined by MTT assay, and the percentage of
cell proliferation was calculated by ~lefinin~ the absorption of cells without tre~tment of
emodin and derivatives as 100%. All delermin~tions were made in triplicate. Results are
3 0 mean + SD.
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Inhibitory Activity Of Emodin ~nd Derivatives
On
Compound Tyrosinephosphorylation Proliferation of
" IC50 (,uM) cells
Group A. C3 Groups:
Emodin 21 S
DK~ 8 > 100 81
DK-III- 19 > 100 45
DK-III-47 39 50
DK-III-48 67 35
DK-III- 13 100 60
DK-III- 11 > 100 > 100
Group B. C6 Groups:
DK-II- I > 100 > 100
DK-II-2 > 100 100
Group C. Cl, C2 Groups:
DK-IV-I >100 >100
Group D. C9 Groups:
DK-V-47 17
DK-V-48 > 100 > 10V
Group E. Cl0 Group:
DK-III-52 >100 >100
The results presented elsewhere in this specification show that emodin effectively
suppresses tyrosine kinase activity of H~R-2/neu, and inhibits, growth of
HER-2/neu-ovclc2~plcssing human breast cancer cells ~see also Zhang et al., 1995). The
inventors also e~mined the effect of emodin and it's derivatives on proliferation of
MDA-MB 453 cells. Cells were treated with or without dirr.,~c,lL concentrations of
emodin and 12 d~,~iv~iv-es at 37~C for 72 hr, then tested using MTT assay. As shown in
Table 2, in the all compounds ~min~l, DK-V-47 is the most effective to against these
cell growth. Emodin has 5-fold less activity than DK-V-47 to inhibit cell proliferation.
Su~plc~ion of Emodin and it's derivatives on tyrosine phorsphorylation of pl85~eU
correlates with the effect these compounds' effect on inhibition of cell proliferation
(Table 2).
"
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These results suggest that CH3 group at C3 position, and OH group at Cl, C6,
and C8 position are very important for m~intzlinin~ inhibitory activity of emodin to
suppress tyrosine phosphorylation of HER-2/neu, and to block growth of
HEI~-2/neu-o~ x~l~ssing breast cancer cells. However, oxygen atom of ketone at Clo
S is replaced by p-ethyl nitro-benzene-metyl-group (DK-V-47) show more effective than
emodin for both inhibitory activity of tyrosine phosphorylation of pl 8SneU and
proliferation of ~IER-2/neu-ovcr~ ssing breast cancer cells. Interestingly, when ethyl
nitro group of DK-V-47 is substituted by nitro group (DK-V-48), the compound show a
little activity, compare with DK-V-47. These results suggest that COCH3 group ofDK-V-47 plays a important role in mzlint~inin~ DK-V-47 inhibitory activity for tyrosine
phosphorylation of p 1 8SneU and proliferation of breast cancer cells.
~X~MpT,F 3
Repression of Autophospholylation and
Transphosphorylation by Emodin in vitro
The results from Exarnple 2 show that phosphotyrosine level of pl85neU is repressed by
emodin. To exarnine whether this rcduction in tyrosine phosphorylation affects the
tyrosine kinase activity of p 1 85ne", the immuno-complex kinase assay was used.
When MDA-MB453 cells were treated with emodin for 24 h. the
autophosphorylation ability of the pl85ne'~ from these cells was inhibited. Further, the
transphosphorylation ability of pl85neU for enolase, an exogenous subskate for tyrosine
kinase, was also significantly decreased compared with L~ ,dl~d cells (FIG. 3A). Hence,
2 5 emodin-treatment of cells results in reduced phosphotyrosine levels in pl 85neU, which in
turn exhibits lower tyrosine kinase activity ~FIG. 1 and FIG. 3A).
To further address whether emodin inhibits intrinsic tyrosine kinase activity of
pl85ne", pl85neU was immuno-precipitated from the untreated MDA-MB453 cells. The3 0 precipitates were treated with different concentrations of emodin and the kinase activity
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measured. The tyrosine kinase activity for both auto- and trans-phosphorylation of
pl85ne" is inhibited by emodin in a dose-dependence manner (FIG. 3B). These results
,, show conclusively that emodin represses the intrinsic tyrosine kinase activity of pl85neU.
Therefore, the reduced phosphotyrosine level in pl85neU by emodin treatment is rmost
likely caused by inhibition of p 1 85neU tyrosine kinase activity.
Since activated HER-2/neu transfolmed 3T3 cells possess high tyrosine kinase of
pl85neZ~ (Stern et al., 1986; Bargmarm and Weinberg, 1988), to address whether emodin
and DK-V-47 can directly inhibit tyrosine kinase activity of activated HER-2/~1eu,
pl85neU was immunc)precipitated from the untreated activated HER-2/neu transforrned
3T3 cells, the precipitates were then treated with varying concentrations of emodin and
DK-V-47, and the kinase activity was measured. The tyrosine kinase activity for both
auto-phosphorylation ability for pl85neU and trans-phosphorylation ability for enolase is
inhibited by emodin and DK-V-47 in a dose-dependent manner ~FIG. 12). Compare vvith
emodin, DK-V-47 is more effective than emodin (FIG. 12). These results conclude that
emodin and DK-V-47 inhibit tyrosine kinase activity of activated pl85neU. Therefore,
the repressed transforrnation of activated HER-2/neu transformed 3T3 is most likely
caused by inhibition of p 1 85neU tyrosine kinase activity.
~X~MPI,~, 4
Effect of Emodin on the Proliferation
of Human Breast Cancer Cells
Since emodin effectively inhibits tyrosine kinase activity of pl85ne" that is critical for
cell growth, it is nece~ry to investigate whether emodin inhibits cell proliferation for
the breast cancer cells that OV~ SS pl85neU. To address this issue, six cell lines were
chosen for further study.
The MDA-MB453, BT-483 and AU-565 cells are neu-ovel~ ssing breast
cancer cell lines as mentioned earlier, the MCF-7 and MDA-MB231 are two human
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breast cancer cell lines expressing basal level of pl85neU. The HBL-100 cell line is
derived from a norrnal human breast tissue transformed by SV40 large T antigen and
expresses basal level of pl 85'te". As shown in FIG. 4, growth of these cells was inhibited
by emodin in a dose-dependent manner, ~ut to different extent. At 40 ~lM concentration
that effectively inhibited tyrosine kinase activity of pl85neU (FIG. 1, FIG. 2 and FIG. 3),
emodin blocked 68%, 72%, and 84% of growth in MDA-MB453, BT-483 and ~U-565
cells, respectively. However, under the same conditions, it only inhibited 37% and 23%
of growth in MCF-7 and MDA-MB 231 cells, respectively.
Emodin had little effect on HBL-l 00 breast cells even up to 80 ~LM concentration.
The differential growth effect was not a~?~ale~lt when the cells were treated with 10 IlM
emodin. This concentration of emodin did not affect tyrosine kinase activity of p 1 85neU.
These results show that emodin preferentially suppresses growth of the
neu-ov~ ssing breast cancer cells and suggests that the differential suppressioneffect is likely through repression of p 1 85neU tyrosine Icinase.
Because emodin at 40 ,uM concentration, inhibited p 1 85ne" tyrosine kinase
activity (FIG. 1, FIG. 2 and FIG. 3) and significantly suppressed growth of breast cancer
cells that OVt;l~;X~ i pl85neU (FIG. 4A), the effect of time course on proliferation of
2 0 MDA-MB453 cells by emodin at 40 ,uM concentration was measured.
Emodin completely inhibited cancer cell growth, while viability of cells was
measured by trypan blue assay, more than 90% of cells were found to be alive (FIG. 4B).
The results show that the mechzlni~m that causes inhibition of cell growth is primarily
2 5 due to repression of proliferation and not induction of cell death under this condition.
In a further study the emodin and DK-V-47 ef~ects on cell proliferation
preferentially for the activated HER-2/neu transformed 3T3 cells was tested. NIH 3T3
parental cell line and its activated HER-2/neu transfor~ned cells were used. As shown in
FIG. 13. the growth of activated HER-2/neu trans~ormed 3T3 cells was inhibited by
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emodin and DK-V-47 in a dose-dependent ma~iner, but to varying extents. ~t 80 ~Mconcentration, emodin and DK-V-47 bLock 55%, and 83% of growth of activated
~, HER-2/neu, respectively. However, under the same condition, both emodin and DK-V-47
had litter effect on parental 3T3 cells. These results indicate that emodin and DK-V-47
preferentially suppress growth of activated HE~-2/neu transformed cells and suggested
that the differential suppression effect occurs through inhibition of pl85neU tyrosine
kinase activity.
F,X~T~Plr,l~. 5
Emodin Induces Differentia~ion of Breast Cancer Cells
As emodin inhibited breast cancer cell growth and no significant cell death wereobserved (FIG. 4B), it is of interest to investigate whether emodin induces
differentiation of breast cancer cells. When MDA-MB453 cells are treated with emodin
(40 ,uM) for 24 h, cells display a flat morphology with larger nuclei and increased
cytoplasm that are characteristic for differentiation (Plowman et al., 1993), compared
~,vith untreated cells, which are moderately adherent, with a rounded morphology.
Maturation of breast cells is characterized by the presence of lipid droplets that
are milk components. Large droplets cont~ining neutral lipid are readily detectable in
emodin-treated cells, in contrast, no large lipid droplets are observed in the untreated
cells. More than 90% of the emodin-treated cells produce large lipid droplets, but only
2-5% untreated cells contain lipid drops and these are much smaller in size. These
results demonstrate that emodin pl~;r~ ially induces differentiation of
neu-over~x~l~s~il,g breast cancer cells, suggesting that the enhanced tyrosine kinase
activity of p 1 85"eU may prevent breast cancer cells from differentiation.
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F,XAIVIP~,F, 6
Effect of Emodin on Transformation of Breast Cancer Cells
One h~llm71rk of the transformed state is the ability of cells to exhibit
anchorage-independent growth. To ~tctPrrnine whether emodin affected this property in
breast cancer cells, cells are seeded into soft agarose and monitored for colony formation
(FIG. 5~. The colony forrnation activity of neu-overexpressing breast cancer cells,
MDA-MB453, BT483 and AU565 in soft agarose cu,~ -g 40 ,uM emodin is
dramatically suppressed. ~owever, under the same condition, the cells, that express
basal level of p 1 85ne", namely MCF-7, MDA-MB23 1 and I IBL- 100 still form significant
number of colonies. The decreased ability to grow in soft agrose for the
neu-ovt;l~;A~l~ssi,lg breast cancer cells does not simply reflect slower proliferation rate
shown in FIG. 4, because additional 3 weeks longer incubation does not increase number
of colonies. Furthermore, no significant change in colony formation activity is observed
when the cells grow in soft agarose cont~ininp 10 ,uM emodin, a concentration that does
not inhibit tyrosine kinase activity of p 1 85neU (F~G. 1 and FIG. 3B).
Taken together, the results indicate that emodin prefelenLially suppresses the
transformation ability of neu-o~ A~les~illg cancer cells and suggest that the
transforrnation s~ ression by emodin may be mediated through its ability to inhibit
tyrosine kinase activity of p 1 85neU.
Whether cells have the ability to exhibit anchorage-independent growth is one
hzlllm~rk to detect cell transformed status. Since emodin can repress hurnan breast cancer
colonies formation in soft agarose (see above and Zhang et al., 19953the inventors
decided to test whether DK-V-47 may also affect this ~lVp~ y in breast cancer, cells
were seeded into soft agarose, and monitored for colony formation. As shown in the F~G.
1 lA, the colony forming activity of H~R-2/neu-o~ ,x~l~,ssing breast cancer
MDA-MB-453 cells in soft agarose cont~ining dirr~ concentrations of DK-V-47 or
emodin was repressed in a dose-dependent manner. However, under the same condition,
3 0 the MCF-7 cells that express basal levels of pl 85neU, still formed a significant number of
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colonies. Interestingly, although DK-V-47 show s~ightly more effective than emodin to
inhibit tyrosine phosphorylation of pl85neU and proliferation of MDA-MB 453 cells
(Table 2), DK-V-47 show much more effective than emodin to repress colony formation
ability of these cells in soft agarose (FIG. I lA). Since activated HER-2/neu transforrned
3T3 cells have high tyrosine kinase activity and have ability to grow in soft agarose
(Stern et al., 1986; Bargmann and Weinberg, 1988). To address whether emodin andDK-V-47 can repress colony formation of activated HER-2/neu transformed 3T3 cells,
activated transformed 3T3 cells were seeded into soft agarose cont~ining different
concentration of emodin and DK-V-47. The colony forrnation of these cells was then
exz1minccl Emodin and DK-V-47 repress colony formation in dose-dependent manner.DK-V-47 is much more effective than emodin (FIG. 11B). These results show that
DK-V-47 as well as emodin inhibits colony formation of activated HER-2/neu
transfo~ed 3T3 cells which may be mediated through suppression of tyrosine kinase
activity of pl85neU in 3T3 cells.
1 5 ~XAI~P I ,F, 7
Effects of h'modin on Tyrosine Phosphorylation and Cell Proliferation
in Human Lung Cancer Cells
This example relates to the neu tyrosine kinase inhibitory activity of emodin in2 0 lung cancer cells. Emodin inhibits neu tyrosine kinase in breast cancer cells as seen in
Examples 2 through 6 above. To test whether it also inhibits neu tyrosine kinase in
human lung cancer cells, the inventors ex~rnine(l the effect of emodin on tyrosine
phosphorylation of pl85neU in NCI-H1435 and NCI-H226 cells.
Cells are treated with emodin at 37~C for 24 h in the absence of serum, then
analyzed for the Levels of pl85neU and its tyrosine phosphorylation. Emodin, at a
concentration of 30 ~lM, significantly reduces the level of tyrosine phosphorylation in
NCI-H 1435 cells (FIG. 6A) but has no effect on pl 85neU level in these cells.
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The inventors have also examined NCI-H460 and its transfectants (Yu et c~l.,
1994). As expected, the parental NCI-H460 and H460.neo control cells express verv low
levels of pl85neU. The other two transfectants express higher levels of pl85neU, their
tyrosine phosphorylation of pl85neU was inhibited by emodin. These results show that
emodin inhibits tyrosine phosphorylation of pl85neU in human lung cancer cells.
l~xAl~lIplr~F 8
Emodin Preferentially Inhibits Proliferation of
neu-overexpressing Human Lung Cancer Cells
Emodin inhibits the tyrosine kinase activity of pl85neU which is critical for cell
growth and preferentially inhibits proliferation neu-overexpressing breast cancer cells
(Examples 2-6 above). To test whether emodin has similar anti-proliferative activity in
lung cancer cells, NCI-H1435, NCI-II226, and NCI-H460 cells and NCI-H460
1 5 transfectants are treated with different concentrations of emodin for 3 days.
As shown in FIG. 7A and FIG. 7B, growth of NCI-H1435, NCI-H226, and
NCI-H460 cells and NCI-H460 cells is inhibited by emodin in a dose-dependent manner,
but to different extent. At the 30 ,uM concentration that effectively inhibits tyrosine
phosphorylation of pl85neU (FIG. 6), emodin blocks 67% of the growth of NCI-H1435
cells ~FIG. 7A). However, under the same conditions, it inhibits only 25% of the growth
of NCI-H226 cells (FIG. 7A). Similar results are obtained in NCI-H460 and its
transfectants. Emodin inhibited 48% and 52% of the growth of H470.eB2 and
H460.eB3, but blocks only 30% and 43% of the growth of parental NCI-H460 cells and
2 5 H460.neo control cells, respectively (FIG. 7B).
These results show that emodin preferentially suppresses growth of the
neu-overex~lessing lung cancer cells and this dirr~ ial suppression effect is me~ tecl
through repression of pl85neU tyrosine phosphorylation.
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~.X~MPLE 9
Emodin Ser ~iti7ef~ neu-Overexpressing Lung
Cancer Cells to Cisplatin, Doxorubicin, and VP16
Since emodin su~ sses tyrosine phosphorylation of neu (FIG. 6A and FIG. 6B),
and ov~.e2~ ssion neu induces resistance to chemotherapeutic drugs (Tsai et al., 1993;
Tsai e f al., 1995), it is imperative to investigate whether emodin sensitizes
neu-overexpressing lung cancer cells to chemoth~ ulic drugs. To explore this
possibility, the inventors ~x~minf~cl the combined effects of emodin with
chemotherapeutic agents on proliferation of NCI-H1435, NCI-H226, and NCI-H460
transfectants.
To identify optimal conditions for these combined tre~1ment~7 the sensitivity ofthese cells to the chemoth~ l d~t;U~iC drugs cisplatin, doxorubicin, and VP 16 was
rlP~rmined. The neu-ov~ t;ssing NCI-H1435 cells show 43-fold more
chemoresistance to cisplatin (FIG. 8A), 8.6-fold more resistance to doxorubicin (FIG.
8B) and 8.5-fold more resistance to VP16 (FIG. 8C) than NCI-H226 cells that express
low levels of neu. The neu-mediated chemoresistance is also evident in the NCI-H460
transfectants but not in the parental NCI-H460 cells, or the control H460.neo cells. The
2 0 H460.eB transfectants show chemorç~i.ct~nee to the ex~mined drugs ranging from
12-fold to 15-fold (FIG. 8A, FIG. 8B and FIG. 8~). These results are con~ t~nt with the
observation that neu ~ e;,~ion induces drug resi~t~nce.
~,X~l~P~,~, 10
2 5 The Combined Effect of Emodin and Chemotherapeutic Agents on
Proliferation of NCI-H1435, NCI-H226, and NCI-H460 Cells
and NCI-H460 Transfectants
The growth of NCI-H1435 cells treated with emodin, cisplatin, doxoruhicin, or
VP16 alone, is inhibited by only 38%, 19%, 24% and 22%, respectively. The
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combinations of emodin with cisplatin, doxorubicin, or VPl6 increases the cell growth
inhibitory activity of these chemotherapeutics to 87%, 92% and l O0%, respectively (FIG.
9A). However, no such synergistic effect is observed in NCI-H226 cells which express
low levels of neu (FIG. 9B). Synergistic effects are also observed in the
neu-ov~,re~lessing H460.eB2 and H460.eB.3 cells (FIG. 9E and FIG. gF). In the
parental NCI-H460 and control H460.neo cells, however, no significant synergistic
anti-proliferative effect is seen ~FIG. 9C and FIG. 9D).
These results show that emodin induces synergistic anti-proliferative activity of
1 0 cispla~in, doxorubicin, and VPl 6 on neu-over~ ssillg lung cancer cells.
F,X~MPT,F,11
Effect of Cisplatin, Doxorubicin and VPl6 alone or in combination with
Emodin on human lung cancer cell colony growth in soft agar
The growth of NCI-H460 cells treated with 30,uM emodin (E), 5,uM cisplatin
(Cis), O.l,uM doxorubicin (DO), or O.l~M VPl6 alone, is inhibited by only 32%, 40,
30% and l ~%, respectively. The combinations of emodin with cisplatin, doxorubicin, or
VPl6 increases the cell growth inhibitory activity of these chemotherapeutics to 70%,
40%, and 52%, respectively (FIG. lOA). Similarly, the growth of H460.neo cells was
inhibited 50% with 30~LM emodin alone, 25% with O.l~M doxorubicin alone, 48% with
5,uM cisplatin alone and 0% with 0.1,uM VPl6. However, the combination of emodinwith cisplatin, doxorubicin and VP16 increases the growth inhibitory activity of these
chemotherapeutics to 72%, 80% and 63%, respectively (FIG. IOB~. The growth of
2 5 H460.eB2 cells was inhibited 55% with 30~M emodin alone, 48% with 0.5~LM
doxorubicin alone, 70% with 75,uM cisplatin alone and 58% with 0.5~M VPl6.
However, the combination of emodin with cisplatin, doxorubicin and VPl6 increases the
growth inhibitory activity of these chemotherapeutics to above 95% in each case (FIG.
lOC). Similarly ~,vith H460.eB3 30~uM emodin inhibited growth by 52%, 0.5~M VPl63 0 inhibited growth by 54% 0.5~1M doxorubicin inhibited growth by 60% and 75,uM
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cisplatin inhibited growth by 65%. However, in combination with emodin the inhibitory
effects of these chemotherapeutic drugs increases to over 95% in each case (FIG. 10D).
The growth of H226 is inhibited by 30% with 30,uM emodin, 30% with 5~LM cisplatin,
25% with O.l,uM doxorubicin and 28% with O.l~M VP16. However, in combination
with emodin the inhibitory activity of these chemotherapeutic drugs increases to 55%,
45% and 50%, respectively (FIG. 10E). The growth of Hl435 is inhibited by 40% with
30,uM emodin, 25% with SOIlM cisplatin, 35% with 1~LM doxorubicin and 32% with
111M VP16. However, in combination with emodin the inhibitor.v activity of thesechemotherapeutic drugs increases to above 95% in each case (FIG. 1 OF).
The results above demon.~tr~ted that emodin inhibited tyrosine phosphorylation of
HER-2/neu in HER-2/neu transfected human lung cancer cells and sensitized the
inhibitory effect of chemotherapeutic agents (cisplatin, doxorubicin, and VP16) on the
proliferation of HER-2/neu-o~,lc~ ssing lung cancer cells (Zhang and Hung, 1996).
Similar results were also obtained from human breast cancer cells (Fig. 16A and FIG.
16~), Colony formation ability of HER-2/neu-o~ x~ressing cancer cells MDA-MB 361(~IG. 16A) in soft agarose contS~ininE~ either emodin, or taxol alone was suppressed by
28% and 31%, respectively. The combinations of emodin with taxol synergisticallyincreased their inhibitory activity to 96% (Fig. 16A). No such synergistic effect was
observed in MDA-MB 435 cells which express low levels of HER-2/neu (Fig. 16B).
These results indicate that emodin induces synergistic inhibitory activity of taxol on
HER-2/neu-ov~ lcx~.lessing breast cancer cells
These results suggested that the tyrosine kinase activity of HER-2/neu is required
2 5 for the chemoresistance of HER-2/neu-o~/~re~ essil1g cancer cells; and that addition of
emodin may improve the efficacy of chemotherapeutic re~imto.n~
HER-2/neu-ove.e~Lc3~ g human breast cancer cells can forrn tumors in athymic
BALB/c nude mice. To test whether emodin suppresses tumor development in mice
bearing HER-2/neu-ovclc,x~ sing cancer cells, tumors were int1~lce~1 by injecl:ing
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HER-2/neu-overexpressing human breast cancer M:[~A-MB 361 cells (5 x 107 cells/0.1
ml/mouse) subcutaneously (s.c.). Then 3 weeks later, when the solid tumor becamepalpable, the mice were treated with either emodin ~40 mg/kg of body weight] or taxol
(10 mg/kg of body weight], or emodin plus taxol [0.2 ml/mouse, intraperitoneal (i.p).
injection] on a 3-days-a-weelc schedule for 8 weeks. Then mice were observed forsurvival up to 10 months. Mice treated with placebo (cremophor: DMSO: saline, 1: 2:
7) contimlPcl to develop tumors and eventually died of tumors between 2-5 months.
Either emodin or taxol alone inhibited HER-2/neu-ovt;l~x~ressing human breast cancer
tumor growth in the nude mice (Fig. 17~), and signif;c~ntly prolonged the life span of
MDA-MB 361 tumor-bearing anim~l~ (Fig. 17B). However, mice treated with emodin
plus taxol. tumor was gradually shrink, finally 2 out of 5 mice are tumor free (Fig. 17A
& 17B). Either emodin alone or emodin plus taxol did not induce mice to lose weight
compared with control mice. These results indicated that emodin synergistically
suppressed growth of HER-2/neu-overexpressing human breast tumor cells, proionged
the life span of HER-2/neu-ov~l~x~lc;ssing tumor mice and suggested that tyrosine kinase
inhibitor emodin may be used as a therapeutic agent for sensitizing
HER-2/neu-overexpressing breast cancers to other HER-2/neu resi.~t~nce drugs, such as
taxol.
To investigate whether the therapeutic effects on tumors in vivo were due to
phosphorylation of HER-2/neu tyrosine kinase was suppressed by emodin, tyrosine
phosphorylation of HER-2/neu in tumors from one mouse in each group were analyzed
by immlmohistochemical staining using antibody against tyrosine phosphorylation or
pl85neU and Western blot. Tyrosine phosphorylation levels in the emodin treated tumor
was almost abolished, con~ed with control tumor however pl85neU protein levels in
the emodin treated tumor was not significantly changed. These results indicated that
emodin suppresses growth of HER-2/neu ov~,.ex~l~.ssing tumor in nude mice through
inhibiting phosphorylation of HER-2/neu tyrosine lcinase.
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FXAMPl.F, 12
Emodin and DK-V-47 repress metastasis-associated properties of activated
~, HER-2/nel~ transformed cells.
Tumor met~t~ei~ is a complex process involving a sequential series of critical
steps (Liotta, 1986; Nicolson, 1988; 1991). During blood-born metastasis, important
steps are the hlll a,L~Lion of tumor cells in the microcirculation and the subsequent
invasion of blood vessel ~Pmçnt membrane. Gelatinase (type IV collagenases) has been
shown to be important participant in the dissolution of the basement membrane collagen
during tumor cell invasion and metastasis (Jayasuriya et al., 1992). The inventors
previous studies demonstrated that activated HER-2/neu transformed 3T3 cells caninduce metastasis in nude mice and have high gel~tin~e activities (Yu and Hung, 1991).
To test whether emodin and DK-V-47 can decrease the secreted protease (gelatinase
collagenase IV) in activated HER-2/neu cells, the effect of emodin and DK-V-47 on
basement membrane-degradation gel~tin~ce in activated HER-2/neu transformed 3T3
cells was examined with zymographic analysis. As shown in FIG. 14A and FIG. 14B,both emodin and DK-V-47 inhibit gelatinolytic activity of the 92 kDa and 68 lcDagel~tinz~ce. The inhibition is enhanced with increasing concentration of either DK-V-47
~FIG. 14B) or emodin (FIG. 14A). DK-V-47, at 10 ,uM concentration shows significant
inhibitory activity for gel~tin~c, however, emodin, at 40 IlM has the similar effete with
DK-V-47 on gel~tin~e activity. These results show that DK-V-47 ;s more effective than
emodin on inhibition of gel~tin:~ce secretion.
Blood-borne m~ n~nt cells must extravasate from the circulation, invade
basement membrane and colonize distant sites to be metastatic. Therefore, cancer cells
invasion is a very important in the met~t~i.c events. Activated HER-2/neu transformed
3T3 cells have been shown to be invasive (Yu and Hung, 1991). To examine whetheremodin and DK-V-47 can abolish the invasive properties of activated HER-2/neu
transforrned cells, in vitro invasion assays were performed to monitor the effect of
emodin and DK-V-47. As shown in the FIG. 15, emodin and DK-V-47 can alnnost
.,
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abolish ability of activated HER-2/neu transformed cells to penetrate the Matrigel layer,
compare with untreated cells (FIG. l S positive control). However, at the 25 ,uMconcentration, DK-V-47 is more effective than emodin to repress invasive ability of'
activated HER-2/neu transformed cells. These results demonstrate that emodin andDK-V47 can completely inhibit the invasive ability of activated HER-2/neu transformed
3T3 cells in vitro. The results that emodin and DK-V-47 significantly decreased the 68
kDa and 92 kDa gelzltin~e production (FIG. 14), and almost abolished ability of
~E~-2/neu-transformed 3T3 cells to invade the Matrigel (FIG. IS), suggest that emodin
and DK-V~7 may ~upple:~S metastasis through repressing of pl85neU tyrosine kinase
1 0 activity.
F,X A l~P~ . 13
In Vivo Prevention of Breast, Lung and Ovaria~ Tumor Development in vivo
by Inhibition of Tyrosine Kinase Activity of HER-~/neu Receptor.
In an initial round of in vivo trials, inventors will use a mouse model of humancancer with the histologic features and metastatic potential resembling tumors seen in
hllm~n~ (Katsumata et al., 1995) and treat these ~nim~l.s with emodin and or emodin-lilce
compound to examine the suppression of tumor development.
These studies are based on the discovery that emodin is an inhibitor for the ne2~
tyrosine kinase receptor and functions as a tumor suppressor for neu-overexpressing
cancer cells (Examples 2-6). The Examples above further show that emodin inhibits the
growth of neu-mediated cancer cells and furthermore sensitizes neu-me~ t~-l cancer
2 5 cells to chemotherapeutic drugs. The current exarnple uses of both neu tyrosine kinase
inhibitors, such as emodin alone or in combination with chemotherapeutic drugs, to
provide a useful preventive and therapeutic regimen for patients with neu-ovelex~l~ssing
cancers.
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Two groups of mice of a suitable cancer model will be treated with doses of
emodin or emodin-like compounds either alone or in combination with anti cancer drugs
starting at 6 weeks of age. Several combinations and concentrations of emodin,
emodin-lilce compounds and anti-cancer drugs will be tested. Control mice will be
r 5 treated with buffer only.
The effect of emodin or emodin-like compounds on the development of breast
tumors will be compared with the control group by e~Rmin~tion of tumor size, pl85neU
tyrosine kinase activity (using IP-western blot analysis) and histopathologic ~x~ ion
(breast tissue will be cut and stained with hematoxylin and eosin) of breast tissue. With
the chemopreventive potential of emodin or emodin-like compounds, it is predicted that,
unlike the control group of mice that develop tumors, the testing group of mice will be
resistant to tumor development.
Breast Cancer Model
In an exemplary study, emodin was tested in mice, at 40 mg/kg, 3 days a week for2 weeks, the mice did not show any weight loss . Although human breast cancer cells
dif~er from murine cancer cells, the inventors will use a similar approach to treat animal
carrying HER-2/neu-ov.,~G~ ssing human breast cancers. Emodin dosage will escalate
2 0 from 40 mg/kg to 60 mg/kg, 80 mg/kg, and lO0 mg/kg. To accomplish this, mice will be
iniected in m.f.p. with 0.1 ml (1-10 x 106) of tumorigenic breast cancer cell suspension
(H~R-2/neu-ov~ essing MDA-MB 361 and BT 474 cells; MDA-MB 435 and
MDA-MB 231 cells which express normal levels of HER-2/neu expression). The mice
will be randomly assigned to untreated control (1 group) and drug-treated groups (4
2 5 groups) of 6 mice/group (total, 30 mice for each cell line). The ~nim~ will be weighed
weekly and tumor volumes will be ~let~ n~cl with calipers using the formulation:volume (mm)3 = width (mm)2 x length (mm)/2 (Giovanella et al., 1982). When palpable
tumor nodules (larger than 2 mm3) can be detected, the tumor-bearing mice will be
treated i.p. with 0.2 ml of placebo [chromophore/DMSO/saline (1:2:7)~ or emodin: at the
3 0 dosage of (a) 40 mg/kg, (b) 60 mg/l~g, (c) 80 mg/kg, (d) 100 mg/kg, 3 days a week. The
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pharrnacokinetics of emodin in rabbits were recently reported: the elimin~tion half-life of
emodin was 227 min~ and oral ~mini~tration of emodin resulted in very low serurnconcentration. Although mice may be different from rabbits in emodin metabolism, i.p.
injection is a preferred method of ~mini~tration because it was effective in thepreliminary studies. The ~nim~lc' tumor volumes will be monitored once a week, and
survival curves ~,vill be drawn. Responses to emodin will be quantified as changes in
tumor volume. Using median values obtained in treated and control ~nim~l~, statistical
significance will be determin~ by the Wilcoxon rank sum test (Dawson-Saunders and
Trapp, l990). Overall toxicity will be measured as percentage of body weight loss and
death. The tumor inhibitory effects of emodin in the two breast tumor systems (i.e.
H~-2/neu ov~,c~lessing or non-ove,expl~s~ g) will be compared. These studies will
thus allow the deterrnination of an optimal dose of emodin with maximum antitumor
activity and lowest toxicity. This sche-~ule and dose will be used for any subsequent
mini~tration.
In the inventors previous studies shown above, both MDA-MB-361 (HER-2/neu
o~/e,~ ssion) and MDA-MB-435 (normal levels of HER-2/neu) cell lines produced
spontaneous metastases after inoculation into m.f.p. These two breast cancer cell lines
will be inoculated into the m.f.p. of mice (15 mice for each line, total of 30 mice). Three
months later, 3 mice inoculated with each cell line will be sacrificed to examine the
formation of metastatic tumors. The rem~inin~ mice inoculated with each cell line will
be divided into 2 groups (6 mice/group). One group will be treated with emodin at
optimal conditions obtained as above, the control group will be given placebo under the
same conditions. Treatment will continue for 3 more months, then the mice will be
2 5 sacrificed and distant metastasis sites will be ex~mined.
Ovarian Cancer Model
In order to obtain mice having human ovarian cancer, nu/nu mice may be given
intraperitoneal injections of, for example, 2 x l06 viable pl85-overexpressing SKOV-3
human ovarian cancer cells. Mice sacrificed 5 days post tre~rm~nt exhibit tumors resulting from such trearm~rlt
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Five days after tre~l~nPnt with the pl85-o~/e~ ressing cells, mice may be
separated into control and experimental groups. One group of mice will be left
untreated. Other groups will be treated. Active compounds may be supplied to a
treated group in phosphate buffer saline. One treated group will be treated with the
buffered saline only. Another treated group may receive treatment with an a~lv~ L~
dosage of emodin or an emodin-like compound alone. A third treated group may be
treated with an a~lol,.iate dosage of an anti-cancer drug alone. A final group may be
treated with an ~)iO~l iate dosage of emodin or an emodin-like compound in
combination with an anti-cancer drug. Tre~tm~nt~ may be given intraperitoneally.
Mice may be ex~min.od for tumor signs and symptoms, and killed when they
appear moribund. Mice treated with the emodin or emodin-like compound plus the
anti-cancer drug will be expected to have a longer survival time.
Small Cell Lung Cancer Model
In order to obtain mice ~,vith the human lung cell cancer, nu/n~ mice may be
given as intratracheal injections of, for example, 2 x 106 viable neu overe~Lessillg
cancer cells from cell line H82. Five days after inoculation, following tumor formation,
mice may be separated into groups to begin ~ .. 1 One group may be treated with an
applopliate dosage of emodin or an emodin-like compound alone, another with an
ap~Lul,liate dosage of an anti-cancer drug alone. A third group may be treated with an
a~,ru~liate dosage of emodin or an emodin-like compound in combination with an
emodin for 3 collse~;u~i~te days, then once a week for two months.
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F.X A l~lPT ,F, 14
Model to test the Effect of Emodin and chemotherapeutic drugs on
HER2/neu overexpressing breast cancer in vitro and in vivo.
Drugs such as paclitaxel, cyclophosphamide, and doxorubicin are currently used
for tre~tment of breast cancer, but these drugs are less effective for patients with
HER-2/neu ov~ ssing breast cancers. The inventors will test paclitaxel for
chemosensitivity of breast cancer cell lines by combined treatment with emodin or
emodin-like compound, because paclitaxel has been used for patients who have advanced
metastatic breast cancer and have failed prior chemotherapy (Chevalier et al., 1995;
Klaassen et al.; Gianna et al., 1994, Chang et al., 1995), and HER-2/neu o~,e.~ essing
is implicated in paclitaxel resistance in vitro (Yu et al., 1996).
Effect of emodin and paclitaxel in combination on growth of breast cancer cells
MDA-MB 361, BT 474, MDA-MB 435, and MDA-MB 231 in vitro. Using a similar
protocol used on lung cancer cells (Zhang and Hung, 1996) for this study. Breast cancer
cells will be first treated with various concentrations of emodin ( 1, 10, 20, 40, 60, 80, and
100 ~uM) alone or paclitaxel (1, 10, and 100 nM, 1, 10, S0, and 100 ,uM) alone for 12, 24,
4~, and 72 hour. The cell growth rate will be determined by the MTT assay (Zhang et al.,
l99S), and the number of viable cells will be determined by trypan-blue staining and
visual counting of samples in a hemocytometer (Zhang et al., 1991). For each time point
and drug treatment, a minimllm of three plates will be counted. In addition, the rate of
DNA synthesis of these cells will be ~let~rTnine~l in parallel plates by the [3H]thymidine
incorporation assay (Zhang et al., 1994). The effect of emodin and paclitaxel on the
ability of forming colonies on soft agar of breast cancer cells will be determinecl by
soft-agar assay (Zhang et al., 1995). The results will be used to establish the
dose-response curves and at various exposure times to critically determine the r~
toxic dose range of the drug and the most efficacious duration of tre~tment for
subsequent studies. Thus, various inhibitory concentrations (ICs~ for each drug can be
determined. To determine whether the combinations exert a synergistic effect, different
ratios of emodin and paclitaxel will be added simultaneously at specific inhibitory levels,
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e g, IC25 + IC25, IC50 + Ic50~ and Icl2 5 + IC75 ( in both combinations). To evaluate
the combined effect of the two drugs, the observed values will be compared to the
predicted values ~c] calculated from the equation [c] = [a] x ~b]/100, where [a] and [b]
indicate survival values with single agents, observed values of less than 70% of predicted
ones will be considered synergistic ~Zhang and Hung, 1996; Hata et al., 1994).
Then, preclinical animal experiments will be used to measure the therapeutic
efficacy of emodirI and emodin-like compounds. The orthotopic breast cancer models
(m.f.p. tumor model, MDA-MB 361, BT 474, MDA-MB 435, and MDA-MB 231 will be
used to induce tumor at m.f.p. of BALB/c nude mice) described above will be used to
test the ability of emodin to sensitize to paclitaxel. When palpable tumor nodules (larger
than 2 mm3) can be detected, the tumor-bearing mice will be given emodin i.p. 3 days a
week, the minimum dose of emodin that maximizes efficacy and minimi7~ toxicity, will
be based on results from Example 13. Paclitaxel has been shown to inhibit tumor growth
in nude mice bearing subcutaneous human mesotheliomas (Lee et al., 199~) and human
lung carcinoma MV 522 xenografts (Kelner et al., 1995) under conditions, respectively,
of i.p. injection of 30 mg/kg, 3 times a week (Lee et al., 1995), and 10 mg/kg, of i.p.
injection 5 days a week for 3 cycles (Kelner et al., 1995). Based on this, the inventors
treat mice bearing breast tumor (6 mice/group) with paclitaxel alone (5, 10, 20, and 30
2 0 mg/kg body weight; for control, the same volume of placebo) in i.p. in~ection 3 times a
week. Then 20%, 50%, and 75% tumor inhibitory doses of paclitaxel will be combined
with emodin (optimal dose obtained from work for Example 13) and ~-lmini~tered i.p. 3
times a week to breast tumor-bearing mice. Tumor size and mouse weight will be
measured once a week, calculation of tumor volume and synergistic effect, and overall
2 5 evaluation of efficacy and toxicity will be done with the methods described for Example
13. From these evaluations the inventors expect the paclitaxel dose required to reduce
turnor volume in the HER-2/neu-ovt~ es~illg ~nim~l~ to be significantly lower when
combined with emodin than the dose of paclitaxel alone, and the HER-2/neu
low-expressing ~nim~ not to be sen~iti7~c~ to paclitaxel by emodin.
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In yet a further the study the inventors propose to determine whether emodin andemodin like compounds prevent breast tumor development by using neu-transgenic mice.
The transgenic mice [transgenic FVB/N-TgN (MMTVneu~ 202] that express the
HER-2/neu oncogene (neuT) in mzlrnm~ry epithelial cells and develop breast tumors at
the mean age of 44 weeks old (comparable to human middle age) are purchased fromJackson Laboratory (Bar Harbor, Marion), and used to ex~minf? whether emodin
suppresses m~mm~ry tumor development. Beginning at 6 weeks of age, these mice (15
mice/group) will be treated with emodin by i.p. injection of 5 different doses (starting at
20 mg/kg of body weight, then rise to 40 mg/kg, 60 mg/kg, 80 mg/kg, and 100 mg/kg,
respectively) 3 times a week. Control transgenic mice will be treated with placebo only.
The effect of emodin on the development of breast tumors will be compared in thetreated and control groups by e~mining tumor size, pl85neU tyrosine lcinase activity
(using immunocomplex assay), tyrosine phosphorylation level of p 1 gSneU (using
immunoprecipitation-western blot analysis~ (Zhang et al., 1995), and histopathological
ex~min~tion (breast tissue will be cut and stained with hematoxylin and eosin).
According to the present invention, the chemopreventive potential of emodin becomes
evident when the tested group will resist breast tumor development.
F'~AMPT,F, 14
2 0 Human Treatment with Emodin-Like Tyrosine Kinase Inhibitors in
Combillation with Anti-cancer Drugs or Alone
This example describes a protocol to facilitate the treatrnent of neu-mediated
cancer using emodin or an emodin-like tyrosine kinase inhibitor alone or in combination
2 5 with anti-cancer drugs.
A patient presenting a neu~ te~ cancer may be treated using the following
protocol. Neu-ove~ ession may be detected using the immunohistochemistry methodsdescribed below. Patients may but need not have received previous chemo- radio- or
gene thc.dpeLILic tre~tment~. Optimally the patient will exhibit adequate bone marrow
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function (defined as peripheral absolute granulocyte count of > 2,000/mm3 and platelet
count of 100, 000/mm3, adequate liver function (bilirubin l.Smg/dl) and adequate renal
function (cre~tinine~ 1.5mg/dl).
Monitori~neu overcxpression ir~ ~ors
The over-e,~ ion of neu is typically m~nit-)red before, during, and after the
therapy. The following assay may be used to monitor neu-overexpression. Sections of 3- to
4 mm thickness of the primary tumors and of the cell block ~p~Lions are cut,
d~ trlil-i7~-1 in xylene, and rehydrated in ~ie~c~nrling grades (100-70%) of ethanol.
Endogenous peroxidase activit;y is blocked with 3% hydrogen peroxide in methanol. After
several washes in distilled water and phosphate-buffered saline, the sections are incubated
with a 1:10 dilution of normal horse serurn to minimi7~ bac~ground st~ining This is
followed by incubation for 1 hr at room temperature with the primary antibody (Ab-3
monoclonal antibody, Oncogene Sciences, Uniondale, NY; 1:100). The peroxidase
st~ining procedure utilizes ABC Elite Kits (Vector Laboratories, Bnrling~me, CA). The
imrnunostaining reactions are vi~ li7.~1 using 3-amino-9-ethylcarbazole as the chromogen.
The sections and/or cytospin p~ ions are stained with toluidine blue and mounted in
permount. Positive and negative control immlmostains are also prepared.
2 0 The sections are reviewed by the pathologist. Two features of the immlmoreaction
will be recorded using a semi ~ LiLalive scale: the relative number of positive cells (0%,
<10%, 10-50%, and > 50%) and the intensity of the reaction (0-3). The pattern ofimmlln~staining (memhr~nous, cytoplasmic) is recorded separately. A tumor is considered
neu positive if any neoplastic cells show cell membrane reactivity. Cytoplasmic st~ininp is
considered non-specific. A breast carcinoma known for its strong positive membrane
st~inin~ will be used as a positive control.
The q~ e measurement of neu immnn-)st:~ining will be performed using
comp-lt~ri7~-1 image analysis with the SAMBA 4000 Cell Image Analysis System (Image
3 0 Products Tntt~rn~tional, Inc., Chantilly, VA) in~egrated with a Windows based software. A
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strong st~ining tumor tissue section will be used as positive control. The primary antibody
will be replaced by an isotype-m~t~ h~d irrelevant antibody to set the negative control
threshold, averaging the results from ten fields.
Proto~ol for the Tr~t~ nt of nell-Mediated C:lncer
A composition of the present invention is typically a-lmini~tered orally or
parenterally in dosage unit formulations COl~t~ g standard, well known non-toxicphysiologically acceptable carriers, adjuvants, and vehicles as desired. The term
parenteral as used herein includes subcutaneous injections, inkavenous, intramuscular,
intra-arterial injection, or infusion techniques.. The emodin or emodin-like compound
may be delivered to the patient before, after or concurrently with the other anti-cancer
agents.
A typical treatment course may comprise about six doses delivered over a 7 to 21day period. Upon election by the clinician the regimen may be continued six doses every
three weeks or on a less fre~uent (monthly, bimonthly, quarterly etc.) basis. Of course,
these are only exemplary times for tre~tment and the skilled practitioner will readily
recognize that many other time-courses are possible.
A major challenge in clinical oncology of neu-mediated cancers is that tumor
cells over-expressing the neu-protooncogene are resistant to chemotheld~eulic treatment.
One goal of the inventors' efforts has been to find ways to improve the efficacy of
chemotherapy. In the context of the present invention, emodin or an emodin-like
compound can be combined with any of a number of conventional chemo~h~ ;c
2 5 regimen~
To kill nel~-ovc~ ing cancer cells using the methods and compositions
described in the present invention one will generally contact a target cell with emodin or
an emodin like tyrosine kinase inhibitor and at least one chemotherapeutic agent (second
3 0 agent), examples of which are described above. These compositions will be provided in
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a combined amount effective to kill or inhibit the proliferation of the cell. This process
may involve cont~ctin~ the cell with emodin or emodin-like compounds and the second
agent at the sarne time. Alternatively, this process may involve contacting the cell with a
single composition or pharmacological forrnulation that includes both agents or by
cont~f~ting the cell with two distinct compositions or forrnulations at the same time,
wherein one composition includes the emodin or emodin-like tyrosine kinase inhibitor
and the other includes the second agent.
~lt~ tively the emodin or emodin-like compound ~ nini.~tration may precede
or follow the delivery of the second agent by intervals ranging from minlltf s to weeks. In
embodiments where the emodin or emodin-like tyrosine kinase inhibitor and the second
compound are applied separately, one would ensure that a significant period of time did
not expire between the time of each delivery, such that the second agent and the emodin
or emodin-like compound would still be able to exert an advantageously combined effect
on the cancer. In such instances, it is contemplated that one would contact the cell with
both agents within about 6 hours to one week of each other and more preferably, within
24-72 hours of each other. In some situations however, it may be desirable to extend the
time period for tre~trnent significantly where several days (2, 3, 4, 5, 6, 7 or more) to
several weeks (1, 2, 3, 4, 5, 6, 7 or more) lapse between respective ~innini~trations.
Regional delivery of emodin or emodin-like tyrosine kinase inhibitors will be anefficient method for delivering a therapeutically effective dose to counteract the clinical
e~e. Likewise, the chemotherapy may be directed to a particular effected region.~lt~rn~tively systemic delivery of either, or both, agent may be ap~lvpliate.
The therapeutic composition of the present invention is ~mini~t~red to the
patient directly at the site of the tumor. This is in essence a topical treatment of the
surface of the cancer. The volume of the composition should usually be sufficient to
ensure that the entire surface of the tumor is contacted by the emodin or emodin like
3 0 compound and second agent.
.
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In one embodiment, ~lmini~tration simply entails in~ection of the therapeutic
composition into the tumor. In another embodiment, a catheter is inserted into the site of
the turnor and the cavity may be continuously perfused for a desired period of time.
Clinical responses may be defined by acceptable measure. For example, a
complete response may be defined by the disappearance of all measurable disease for at
least a month. Whereas a partial response may be defined by a 50% or greater reduction
of the sum of the products of perpendicular diameters of all evaluable tumor nodules or
at least 1 month with no tumor sites showing enlargement. Similarly, a mixed response
mav be defined by a reduction of the product of perpendicular diameters of all
measurable lesions by 50% or greater with progression in one or more sites.
Of course, the above-described treatment regimes may be altered in accordance
with the knowledge gained from clinical trials such as those described in Example 14.
Those of skill in the art will be able to take the information disclosed in this specification
and optimize treatment regimes based on the clinical trials described in the specification.
F'X~MPLE 16
2 o Clinical Trials of the Use of Tyrosine Kinase Inhibitors in Combination with
Anti-cancer Drugs or Alone in Treating Neu-Me~ te~l Cancer
This exarnple is concerned with the development of human tre~tment protocols
using the emodin and emodin-like compound alone or in combination with anti-cancer
2 5 drugs. Emodin or emodin-like compounds and anti-cancer drug treatment will be of use
in the clinical tre~tm~nt of various neu-ovclc;~ ;ssing cancers in which transformed or
cancerous cells play a role. ~uch tre~tment will be particularly useful tools in anti-tumor
therapy, for example, in treating patients with ovarian, ~reast and lung cancers that are
me~ teA by neu over-expression and resistant to conventional chemotherapeutic
3 o regimens.
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The various elements of conducting a clinical trial, including patient tre~tm~ntand monitoring, will be known to those of skill in the art in light of the present
disclosure. The following information is being presented as a general guideline for use
in establishing emodin or emodin-like compounds alone or in combinations with
anti-cancer drugs in clinical trials.
Patients with advanced, metastatic breast and/or epithelial ovarian carcinoma
chosen for clinical study will typically have failed to respond to at least one course of
conventional therapy. Measurable disease is not required, however the patient must have
easily accessible pleural effusion andlor ascites. Further the patients must carry tumors that
O~ X~IeSs neu oncoprotein. O~ ~ -cx~-es~ion may be defined as grade 2 or 3 staining by
immunohistor.h~mi~try as described above. In an exemplary clinical protocol, patients
may undergo pl~rem~nt of a Tenckhoff c~th~ter, or other suitable device, in the pleural or
peritoneal cavity and undergo serial sampling of pleural/peritoneal effusion. Typically, one
will wish to determine the absence of known loculation of the pleural or peritoneal cavity,
creatinine levels that are below 2 mg/dl, and bilirubin levels that are below 2 mg/dl. The
patient should exhibit a normal coagulation profile.
In regard to the emodin or emodin-like compounds and other anti-cancer drug
sl~lmini.ctration, a Tenckhoff catheter, or alternative device may be placed in the pleural
cavity or in the peritoneal cavity, unless such a device is already in place from prior
surgery. A sarnple of pleural or peritoneal fluid can be obtained, so that baselline
cellularity, cytology, LDH, and a~loplidLe markers in the fluid (CEA, CA15-3, CA 125,
2 5 pl 85) and in the cells (ElA, pl 85~ may be ~çssecl and recorded
In the same procedure, emodin or emodin-like compourld may be ~1minietered
alone or in combination with the anti-cancer drug. The ~imini~t~ation may be in the
pleural/peritoneal cavity, directly into the tumor, or in a systemic manner. The starting
3 0 dose may be O.Smg/kg body weight. Three patients may be treated at each dose level in the
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absence of grade > 3 toxicity. Dose escalation may be done by 100% increments (O.Smg,
lmg, 2mg, 4mg) until drug related grade 2 toxicity is dt~tecte(1 Thereafter dose escalation
may proceed by 25% increments. The ~1mini~t~red dose may be fractionated equally into
two infusions, separated by six hours if the combined endotoxin levels determined for the
lot of emodin or emodin-like compound and the lot of anti-cancer drug exceed 5EUAcg for
any given patient.
The emodin or emodin-like compound and anti-cancer drug combination may be
~lmini~tered over a short infusion time or at a steady rate of infusion over a 7 to 21 day
period. The emodin and/or emodin-like compound infusion may be ~rlmini~t~red alone
or in combination with the anti-cancer drug. The infusion given at any dose level will be
dependent upon the toxicity achieved after each. Hence, if Grade II toxicity was reached
after any single infusion, or at a particular period of time for a steady rate infusion,
further doses should be withheld or the steady rate infusion stopped unless toxicity
improved. Increasing doses of emodin and/or emodin-like compound alone or in
combination with an anti-cancer drug will be ~tlmini~tered to groups of patients until
approximately 60% of patients showed unacceptable Grade III or IV toxicity in any
category. Doses that are 2/3 of this value could be defined as the safe dose.
2 0 Physical e~c~min~tion, tumor measule.llents, and laboratory tests should, of
course, be performed before treatment and at intervals of about 3-4 weelcs later.
Laboratory studies should include CBC, dirr~;lell~ial and platelet count, urinalysis,
SMA-12-100 (liver and renal function tests), coagulation profile, and any other ap~lo~l;dl~
~hl?miclTy studies to ~l~otermine the extent of disease, or ~l~tprmine the cause of existing
symptoms. Also a~lopliate biological markers in serum should be monitored e.g. CEA,
CA 15-3, pl85 and pl85neU tyrosine phosphorylation for breast cancer, and CA 125, pl85
tyrosine phosphorylation for ovarian cancer
To monitor disease course and evaluate the anti-tumor responses, it is
contemplated that the patients should be examined for ~lo~l;ate tumor markers every 4
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weeks, if initially abnormal. with twice weekly CBC, differential and platelet count for the
4 weeks; then, if no myelosuppression has been observed, weekly. If any patient has
A prolonged myelo~u~ s~ion, a bone marrow ~x~min~tion is advised to rule out the
possibility of tumor invasion of the marrow as the cause of pancytopenia. Coagulalion
~ 5 profile shall be obtained every 4 weeks. An SMA-12-100 shall be p~l~oLll~ed weekly.
Pleural/peritoneal effilsion may be sampled 72 hours after the first dose, weekly thereafter
for the first two courses, then every 4 weeks until progression or off study. Cellularity,
cytology, LDH, and a~lu~l;ate markers in the fluid (CEA, CA15-3, CA 125, pl85
tyrosine phosphorylation) and in the cells (pl85 tyrosine phosphorylation) may be
assessed. For an example of an evaluation profile, see Table 3. When measurable disease is
present, tumor measurements are to be recorded every 4 weeks. A~lo~.iale radiological
studies should be repeated every 8 weeks to evaluate tumor response. Spirometry ~nd
DLCO may be repeated 4 and 8 weeks after initiation of therapy and at the time study
participation ends. An urinalysis may be performed every 4 weeks.
Clinical responses may be defined by acceptable measure. For example, a
complete response may be defined by the disappearance of all measurable disease for at
least a month. Whereas a partial response may be defined by a 50% or greater reduction
of the sum of the products of perpendicular diameters of all evaluable tumor nodules or
2 o at least I month with no tumor sites showing enlargement. Similarly, a mixed response
may be defined by a reduction of the product of perpendicular diameters of all
measurable lesions by 50% or greater with progression in one or more sites.
SUBST~TUTE SHEET (RULE 26)
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Table 3
EVALUATIONS BEFORE AND DURING THERAPY
EVALUATIONS PRE-STUD TWICE WEEKLY EVERY 4 EVERY 8
Y WEEKLY WEEKS WEEKS
History X X
Physical X X
Tumor Mcz~u,~",dnl~ X X
CBC X X' X
Dir~ "lial X Xl X
Platelet Count X Xl X
SMA12-100 (SGPT, Alkaline X X
Pl,os~ alase, ~ilirubin,
AlblTotal Protein)
Co~yuldlion Profile X X
Serum Tumor markers ~CEA, X X3
CA15-3, CA-125, Her-2/neu
tyrosine ph~r':~rylatinn)
Urinalysis X X
X-rays:
chest X X4
others X X
PleurallP~,ilu"eal Fluids: X X5 X
(cellularity, cytology, LDH,
tumor markers, level of
HER-21neu tyrosine
, ' ~ " ' rylalion)
Spirometry and DLCO X X5 X6
For the first 4 weeks, then weekly, if no I~ u~ Jl tssion is observed.
As indicated by the patient's col1diliol1.
Repeated every 4 weeks if initially dbl)Grllldl.
4 For patients with pleural effusion, chest X-rays may be pe~ ~u. I,led at 72 hours after first dose, then
prior to each ll~allllt lll admin;~lldliom
Fluids may be assessed 72 hours after the first dose, weekly for the first two courses and then
every 4 weeks Ih~.~a~lel.
6 Four and eight weeks after initiation of therapy.
~ * *
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All of the compositions and methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present disclosure. While the
compositions and methods of this invention have been described in terms of ~ler~ d
embo~iime.nt.c, it will be ~ lt to those of skill in the art that variations may be applied
to the composition, methods and in the steps or in the sequence of steps of the method
described herein without departing from the concept, spirit and scope of the invention.
More specifically, it will be a~ nt that certain agents which are both chemically and
physiologically related may be substituted for the agents described herein while the same
or similar results would be achieved. All such similar substitutes and modifications
~pal~nt to those skilled in the art are deemed to be within the spirit, scope and concept
of the invention as defined by the appended claims.
1 5 REFEI~F,NCE~S
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