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Cancer Patent Abstract
Improvements in methods of treating cancer with weakly basic anti-cancer
compounds are provided. In one aspect, the invention provides an
improvement in a method of treating cancer cells whose extracellular
environment contains 1 8 mM urea, by exposing the cells to a weakly
basic anti-cancer compound which is effective in inhibiting the
growth of the cells. The improvement includes (a) exposing the cells
to a urease enzyme composition and, (b) by step (a), reducing the
amount of anti-cancer compound required to produce a given extent
of inhibition in the growth of the cells when the cells are exposed
to the anti-cancer agent. Methods of potentiating the specific therapeutic
activity of a weakly basic anti-cancer compound in the treatment
of a given mammalian cancer which is responsive to the compound
are provided as are pharmaceutical compositions for use in intravenous
administration to a subject are also provided.
Cancer Patent Claims
It is claimed:
1. In a method of treating cancer cells whose extracellular environment
contains 1 8 mM urea, by exposing said cells to a weakly basic anti-cancer
compound which is effective in inhibiting the growth of said cells,
an improvement comprising (a) exposing the cells to a composition
containing a urease enzyme, and (b) by step (a), reducing the amount
of said anti-cancer compound required to produce a given extent
of inhibition in the growth of said cells, when the cells are exposed
to the anti-cancer agent.
2. The improvement of claim 1, wherein said exposing is effective
to raise the pH of the extracellular environment of the cancer cells
by at least 0.1 pH unit.
3. The improvement of claim 2, wherein said anti-cancer compound
is selected from the group consisting of doxorubicin, daunorubicin,
mitoxantrone, epirubicin, mitomycin, bleomycin, a vinca alkaloid,
an alkylating agent, or an antineoplastic purine and pyrimidine
derivative.
4. The improvement of claim 2, wherein the amount of said anti-cancer
compound required to achieve the same extent of cell-growth inhibition
is between about 2-fold and 5-fold less than in the absence of step
(a).
5. The improvement of claim 2, wherein said composition includes
a targeting moiety attached to said urease enzyme and selected from
the group consisting of an antibody directed against a tumor antigen,
an anti-hCG antibody, and a ligand capable of binding specifically
to a cancer-cell surface receptor.
6. The improvement of claim 5, wherein said targeting moiety is
a single-chain antibody.
7. The improvement of claim 2, in a method for treating a cancer
in a mammalian subject, by administration of such anti-cancer compound
to the subject, wherein step (a) includes administering to the subject,
a urease composition effective to localize at the site of the cancer
in the subject.
8. The improvement of claim 7, wherein said urease composition
is administered intravenously in an amount of between 25 and 2000
pmoles urease enzyme/kg subject body weight, at least 24 hours prior
to administration of the anti-cancer compound.
9. The improvement of claim 8, wherein said urease composition
is administered by IV drip and the composition includes a urease
inhibitor selected from the group consisting of a hydroxamic acid
derivative and other urea analogs, in an amount effective inhibit
the activity of the urease enzyme at the initial concentration of
the urease composition.
10. The improvement of claim 7, for use in treating a solid tumor,
wherein said urease composition is administered directly in the
tumor in an amount effective to raise the pH total of the extracellular
fluid of said tumor, as evidenced by detectable change in a pH indicator
present in the tumor extracellular fluid.
11. A method of treating a cancer that is responsive to a selected
weakly basic anti-tumor compound in mammalian subject, comprising:
administering to the subject, such weakly basic anti-cancer compound
and an amount of a urease enzyme composition sufficient to potentiate
the therapeutic effect of the compound with respect to the therapeutic
effect obtained in the absence of said urease composition administration.
12. The method of claim 11, which includes administering said urease
composition at least 24 hours prior to administering the anti-cancer
compound.
13. The method of claim 11, which includes co-administering the
anti-cancer compound and the urease composition.
14. The method of claim 11, wherein urease composition is administered
in an amount effective to raise the pH of the extracellular environment
of the cancer cells by at least 0.1 pH unit.
15. The method of claim 11, wherein administration of the urease
composition is effective to reduce the amount of said anti-cancer
compound required to achieve the same extent of cell-growth inhibition
by a factor of between 2 and 5.
16. The method of claim 11, wherein said anti-cancer compound is
selected from the group consisting of doxorubicin, daunorubicin,
mitoxantrone, epirubicin, mitomycin, bleomycin, a vinca alkaloid,
an alkylating agent, or an antineoplastic purine or pyrimidine derivative.
17. The method of claim 11, wherein said urease composition includes
a targeting moiety attached to said urease and selected from the
group consisting of an antibody directed against a tumor antigen,
an anti-hCG antibody, and a ligand capable of binding specifically
to an cancer-cell surface receptor.
18. The method of claim 11, wherein said urease composition is
administered by IV drip in an amount of between 25 and 2000 pmoles
urease enzyme/kg subject body weight, and the composition includes
a urease inhibitor selected from the group consisting of a hydroxamic
acid derivative and other urea analogs, in an amount effective to
inhibit the activity of the urease enzyme at the initial concentration
of the urease composition.
19. In a subject having a solid tumor a method of enhancing the
therapeutic efficacy of a weakly basic anti-tumor compound whose
effectiveness is reduced by a higher intracellular/lower extracellular
pH gradient in a solid tumor, comprising administering to the subject
receiving said anti-tumor compound, an amount of urease effective
to reduce or reverse the higher intracellular/lower extracellular
pH gradient in a solid tumor.
20. The method of claim 19, wherein said anti-tumor compound is
selected from the group consisting of doxorubicin, dauorubicin,
mitoxanthrone, epirubicin, mitomycin, bleomycin, vinca alkaloids,
alkylating agents and antineoplastic purine and pyrimidine derivatives.
21. The method of claim 19, wherein said administering is effective
to raise the extracellular fluid of the tumor of at least pH 7.2.
22. The method of claim 19, wherein said administering includes
injecting urease directly into the subject's tumor.
23. The method of claim 19, wherein said urease is administered
parenterally other than by direct injection in a composition that
includes a chemical entity effective to enhance the delivery of
the enzyme to a solid tumor.
24. The method of claim 20, wherein the vinca alkaloid is selected
from the group consisting of vinblastine and vincristine.
25. The method of claim 20, wherein the alkylating agent is selected
from the group consisting of cyclophosphamide and mechlorethamine
hydrochloride.
Cancer Patent Description
FIELD OF THE INVENTION
The present invention relates to anticancer therapeutic methods
employing urease in combination with a weakly basic anti-cancer
compound.
BACKGROUND OF THE INVENTION
Cancer accounts for one-fifth of the total mortality in the United
States, and is the second leading cause of death. Cancer is typically
characterized by the uncontrolled division of a population of cells.
This uncontrolled division may involve blood cells, such as various
types of lymphomas, or cells that aggregate in or are native to
a particular tissue or organ, e.g., solid tumors, such as secondary
or primary tumors of the breast, lung, liver, esophagus, stomach,
intestines, brain, bone, or prostate.
A variety of treatment modalities have been proposed for cancer
therapy. These generally include surgical resection of solid tumors,
treatment with radiation, such as x-ray, chemotherapy, immune therapy,
and gene therapy. The type(s) of therapy that are selected for a
given cancer will depend on such factors as patient age, degree
of localization of the cancer, and the type and stage of the cancer.
Often the therapy will involve a combination of two or more modalities,
such as x-ray therapy in combination with chemotherapy, or with
immunotherapy in combination with chemotherapy.
A large number of chemotherapeutic compounds and compositions and
strategies have been employed in treating cancers. Many anti-neoplastic
compounds are designed to disrupt replication in rapidly dividing
cells, or to inhibit a key metabolic link in actively proliferating
cells. Although such approaches have met with levels of success
in certain types of cancers, or cancers at certain stages, chemotherapy
is generally associated with unpleasant to debilitating side effects,
such as malaise, nausea, loss of appetite, alopecia, and anemia,
and in the extreme, loss of immune function and/or loss of digestive
activity. Further, compounds which act at the level of cell replication,
either by introducing nucleotide analogs into dividing cells, or
by disrupting normal replication, have the potential of introducing
widespread genetic mutations in normal cells in the subject. In
addition, cancer cells may develop resistance to many types of anti-cancer
agents, either by limiting uptake of the agent into the cells, or
by altering the metabolism of the agent within the cells.
In response to these limitations, attempts to modify chemotherapeutic
agents to reduce their side effects, overcome problems of resistance,
or improve their targeting to selected tumor sites have been developed.
While these efforts have yielded improved therapeutic results in
some cases, there remains a need to provide an improved chemotherapeutic
agent and method. In particular, such an agent and method should
be effective in killing or inhibiting the growth of cancer cells,
should be relatively non-toxic at therapeutically effective doses,
and preferably deliverable in a form that allows direct introduction
into a tumor or selective targeting to tumors.
SUMMARY OF THE INVENTION
It has been discovered that the efficacy of a weakly basic anti-cancer
compound can be enhanced when cancer cells, a patient or other subject
is also treated with urease. That is, the amount of the weakly basic
anti-cancer compound required to achieve a specified amount of growth
inhibition in the presence of urease is less than in the absence
of urease. Accordingly, the specific therapeutic efficacy of a given
amount of anti-cancer drug in a mammalian subject can be enhanced
(potentiated), by co-treating the subject with urease, typically
by pretreating the subject with urease. Accordingly, improvements
in a method of treating cancer cells are provided. Methods of potentiating
the specific therapeutic activity of a weakly basic anti-cancer
compound are provided, as are pharmaceutical compositions for use
in intravenous administration of a subject.
In one aspect of the invention, improvements in a method of treating
cancer cells whose extracellular environment contains 1 8 mM urea
by exposing the cells to a weakly basic anti-cancer compound which
is effective in inhibiting the growth of the cells is provided.
In a general embodiment, the improvement includes (a) exposing the
cells to a urease enzyme composition and (b) by step (a) reducing
the amount of anti-cancer compound required to produce a given extent
of inhibition in the growth of the cells when the cells are exposed
to the anti-cancer agent.
The amount of urease to which the cells are exposed is preferably
sufficient to raise the pH of the extracellular environment of the
cells by at least about 0.1 pH units, typically between 0.1 to 0.5
or more pH units. The amount of anti-cancer compound required to
achieve the same extent of cell-growth inhibition may be between
about 2-fold and 5-fold less than in the absence of step the urease
administration.
Exemplary anti-cancer agents include doxorubicin, daunorubicin,
mitoxantrone, epirubicin, mitomycin, bleomycin, a vinca alkaloid,
an alkylating agent, and antineoplastic purine and pyrimidine derivatives.
The urease composition may include a targeting moiety, such as
an antibody directed against a tumor-specific antigen, an anti-hCG
antibody, and a ligand capable of binding specifically to a cancer-cell
surface receptor. The targeting moiety may be covalently attached
to the urease enzyme. One exemplary targeting moiety is a single-chain
antibody, such as a single-chain antibody derived from llama germline.
For use in treating a cancer in a mammalian subject, by administrating
the anti-cancer compound to the subject, the urease composition
administered to the subject is effective to localize at the site
of the cancer into the subject. The urease composition may be administered
intravenously, e.g., in an amount of between 25 and 2000 pmoles
urease enzyme/kg subject body weight, at least 24 hours prior to
administration of the anti-cancer compound. A urease composition
administered by IV drip may include a urease inhibitor, in an amount
of at least about 6 moles inhibitor/mole urease enzyme.
Alternatively, for use in treating a solid tumor, the urease composition
may be administered directly in the tumor in an amount effective
to raise the pH total of the extracellular fluid of the tumor, as
evidenced by detectable change in a pH indicator present in the
tumor extracellular fluid.
In another aspect, the invention includes a method of treating
a cancer that is responsive to a selected weakly basic anti-tumor
compound in mammalian subject. The method includes administering
to the subject, such weakly basic anti-cancer compound, and an amount
of urease sufficient to potentiate the therapeutic effect of the
compound with respect to the therapeutic effect obtained in the
absence of said urease administration. The urease may be administered
at least 24 hours prior to administering the anti-cancer compound,
allowing clearance of at least a significant portion of the urease,
prior to administering the anti-cancer compound. The amount of urease
administered may be effective to raise the pH of the extracellular
environment of the cancer cells by at least 0.1 pH unit. The amount
of urease administered may be effective, for example, to reduce
the amount of said anti-cancer compound required to achieve the
same extent of cell-growth inhibition by a factor of between 2 and
5.
In still another aspect, the invention includes a pharmaceutical
composition for use in intravenous administration to a subject.
The composition may include at least about 20 Units urease enzyme/ml;
a urease inhibitor at a concentration effective to inhibit the urease
enzyme, and a physiological carrier suitable for administering the
composition intravenously. In one embodiment, where the enzyme has
six enzymatic subunits, the molar ratio of inhibitor to enzyme is
preferably at least about 6:1.
The urease enzyme may be covalently linked to a targeting agent
selected from the group consisting of an antibody directed against
a tumor antigen, an anti-hCG antibody, and a ligand capable of binding
specifically to a cancer-cell surface receptor.
The urease inhibitor may be a hydroxamic acid derivative or other
urea analog.
These and other objects and advantages of the present invention
will be apparent from the description herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A 1D illustrate the steps of the urease reaction. Urea is
cleaved by urease to produce one molecule of ammonia and one of
carbamate (A). Carbamate spontaneously decomposes to ammonia and
carbonic acid (B). The carbonic acid equilibrates in water (C),
as do the two molecules of ammonia, which become protonated to yield
ammonium and hydroxide ions (D). The reaction results in a rise
in the pH of the reaction environment.
FIG. 2 shows the mass spectrometry profile of a crude sample containing
urease prepared in accordance with one embodiment of the invention.
FIG. 3 illustrates the affinity purification profiles of urease
during various stages of the purification process, in accordance
with another embodiment of the invention.
FIG. 4 illustrates the purification of E-coil-.alpha.hEGFR IgG
conjugate by a protein-G column prepared according to one embodiment
of the invention.
FIG. 5 shows the antibody titer of purified E-coil-.alpha.hEGFR
IgG conjugate prepared according to one embodiment of the invention
as determined by immobilized K-coil ELISA.
FIG. 6A is a graph showing a dose-response curve of urea on the
viability of A549 (.tangle-solidup.) and MDA-MB-231 (.circle-solid.)
cells. Cells were incubated in 0 40 mM urea, treated with 2 U/ml
of urease and incubated at 37.degree. C. for 2 hours as more fully
described in Example 7. Viability of the treated cells began to
drop as the urea level increased. Urea alone has no effects on A549
(.DELTA.) and MDA-MB-231 (.largecircle.) controls.
FIG. 6B is a graph showing a dose-response curve of urease on the
viability of A549 (.tangle-solidup.) and MDA-MB-231 (.circle-solid.)
cells. Cells were incubated in 20 mM urea and treated with 2 U/ml
of urease for 2 hours as described in Example 7. A549 (.tangle-solidup.)
were more susceptible to urease than MDA-MB-231 (.circle-solid.)
cells.
FIG. 6C is a graph showing total ammonium ion as a function of
urea treatment in pooled incubation buffer collected from A549 cells
treated with urease as described for FIG. 6A and as more fully described
in Example 8. Hydrolysis of urea by urease (.box-solid.) caused
an increase in ammonium content as compared to the control (.quadrature.).
Values are means.+-.S.D. of 4 replicates from 3 experiments.
FIG. 6D is a graph of pH as a function of urea treatment in pooled
incubation buffer collected from A549 cells treated with urease
as described for FIG. 6A and as more fully described in Example
8. Hydrolysis of urea by urease (.box-solid.) caused an increase
in pH as compared to the control (.quadrature.). Values are means.+-.S.D.
of 4 replicates from 3 experiments.
FIGS. 7A 7F are graphs depicting the protective effects of acetohydroxamic
acid (AHA) on urease cytotoxicity as described in Example 9. (A)
A549 cells (.tangle-solidup.) and (B) MDA-MB-231 cells (.circle-solid.)
treated with 2 U/ml of urease were protected from the cytotoxic
effects by addition of acetohydroxamic acid to the incubation buffer.
AHA alone at concentrations up to 6 mM was not toxic to both cell
lines (no urease controls: .DELTA., A549; .largecircle., MDA-MB-231).
Complete protection was observed at dose .gtoreq.2 mM. (C) AHA inhibited
ammonium production by urease (.box-solid.), which corresponds to
an increase in survival rate of both cell lines as shown in (A)
and (B). Higher amount of AHA (6 mM) can reduce the ammonium level
close to that of control (.quadrature.). Values are means.+-.S.D.
of 4 replicates from 3 experiments. (D) AHA inhibited ammonium production
by urease at indicated urea concentrations; (E) A549 cells; or MDA-MB-231
cells incubated in the indicated amounts of urea and treated with
2 U/ml urease were protected from the cytotoxic effects of urease
by addition of acetohydroxamic acid to the incubation buffer.
FIGS. 8A 8B are graphs which depict the growth inhibitor effects
of urease on tumor cell line xenografts as described in Example
10. (A) urease inhibits the growth of established MCF-7 xenografts.
The breast tumor stopped to grow after the second injection of high-dose
of urease (10 U/injection, solid bars) on day 9 as compared to the
controls (open bars). Time of intratumoral injections are indicated
by .DELTA. below the x-axis. (B) effects of multiple low-dose (1
U/injection, hatched bars) and medium-dose (4 U/injection, solid
bars) injections of urease on established A549 xenografts. Intratumoral
injections were performed on days 5, 7, 9, 11 and 13 (.DELTA.).
Delay of tumor growth was observed from days 17 onwards as compared
to the controls (open bars). Significance was determined using the
two-tailed unpaired Student's t test: *P<0.05 and **P<0.005.
FIGS. 9A 9B are graphs depicting the effects of urease on the cytotoxicity
of weakly basic anticancer drugs as described in Example 11. (A)
lung tumor A549 and (B) breast tumor MDA-MB-231 incubated in 0,
2 or 8 mM urea, were treated with 2 U/ml of urease, and either 50
.mu.M of doxorubicin or 100 .mu.M of vinblastine at pH 6.8 overnight.
The antitumor efficacies of the two compounds were enhanced at the
presence of urease (solid bars) and urea as compared to the control
(open bars). The solid circle (.circle-solid.) indicates the pH
of urease-treated group measured after treatment. Values are means.+-.S.D.
of 4 replicates from 3 experiments.
FIGS. 10A 10B are graphs showing the effects of urease on the cytotoxicity
of weakly basic anticancer drugs as described in Example 11. Lung
tumor A549 (A) and breast tumor MDA-MB-231 (B) were incubated in
urea and treated with DOS47 (2 U/ml), and either Fluorouracil (13.3
mM) or Mitoxantrone (5 .mu.M) at pH 6.8 overnight. The enhanced
anticancer effect (solid bar) of Mitoxantrone is only observed in
MDA-MB-231 but not in A549 cells. DOS47 also enhances the anticancer
effects of Fluorouracil in A549 but not in MDA-MB-231. The solid
circle (.circle-solid.) denotes the pH of DOS47 group measured after
treatment. Values are means.+-.S.D. of 4 replicates from 3 different
experiments.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides improvements in selected methods
of treating cancer cells, methods of potentiating the specific therapeutic
activity of a weakly basic anti-cancer compound, and pharmaceutical
compositions for inhibiting the growth of cancer cell, e.g., in
a mammalian patient. It has been discovered that the pharmaceutical
efficacy of a weakly basic anti-cancer compound can be enhanced
when a mammalian subject, such as a human subject, is also treated,
preferably pretreated with urease.
I. Definitions
Unless otherwise indicated, all technical and scientific terms
used herein have the same meaning as they would to one skilled in
the art of the present invention.
The term "urease" refers to an enzyme having the enzymatic
activity of a urea amidohydrolase (E.C. 3.5.1.5), either naturally
occurring or obtained by e.g., recombinant nucleic acid techniques
and/or chemical synthesis. Urease also includes fusion proteins
comprising the entire urease, subunits, or fragments thereof, and/or
urease with amino acid substitutions, deletions or additions that
preserve the urea amidohydrolase activity of the polypeptide. A
truncated urease sequence as used herein is a fragment of urease
that is free from a portion of the intact urease sequence beginning
at either the amino or carboxy terminus of urease. Methods for isolating
native urease, for synthesizing urease recombinantly, and for identifying
active fragments and modified urease polypeptides are given below.
The term "cancer" is meant to refer to an abnormal cell
or cells, or a mass of tissue. The growth of these cells or tissues
exceeds and is uncoordinated with that of the normal tissues or
cells, and persists in the same excessive manner after cessation
of the stimuli which evoked the change. These neoplastic tissues
or cells show a lack of structural organization and coordination
relative to normal tissues or cells which may result in a mass of
tissues or cells which can be either benign or malignant. As used
herein, cancer includes any neoplasm. This includes, but is not
limited to, melanoma, adenocarcinoma, malignant glioma, prostatic
carcinoma, kidney carcinoma, bladder carcinoma, pancreatic carcinoma,
thyroid carcinoma, lung carcinoma, colon carcinoma, rectal carcinoma,
brain carcinoma, liver carcinoma, breast carcinoma, ovary carcinoma,
and the like.
A "tumor" or "solid tumor" refers to a cohesive
mass of cancer cells, including but not limited to semi-solid and
solid tumors, solid tumor metastases, angiofibromas, retrolental
fibroplasia, hemangiomas, and Karposi's sarcoma.
As used herein, the term "targeting moiety" refers to
a molecule that is associated with urease and which is effective
to promote localization of urease at cancer cells, e.g., within
a solid tumor. In one general embodiment, the targeting moiety is
a binding molecule covalently attached to urease and capable of
binding a receptor, an oligonucleotide, an enzymatic substrate,
an antigenic determinant, or other binding site present on or in
the target cell or cell population. An exemplary targeting moiety
is an antibody, including antibody fragments and constructs, such
as a single-chain antibody, and/or an antibody derived from human
germline. In another general embodiment, the targeting agent is
a long-circulating particle, such as PEG-coated liposomes capable
of localizing at tumor sites by extravasation, and containing encapsulated
or surface bound urease.
As used herein, the term "inhibits growth of cancer cells"
or "inhibiting growth of cancer cells" refers to any slowing
of the rate of cancer cell proliferation and/or migration, arrest
of cancer cell proliferation and/or migration, or killing of cancer
cells, such that the rate of cancer cell growth is reduced in comparison
with the observed or predicted rate of growth of an untreated control
cancer cell. The term "inhibits growth" can also refer
to a reduction in size or disappearance of a cancer cell or tumor,
as well as to a reduction in its metastatic potential. Preferably,
such an inhibition at the cellular level may reduce the size, deter
the growth, reduce the aggressiveness, or prevent or inhibit metastasis
of a cancer in a patient. Those skilled in the art can readily determine,
by any of a variety of suitable indicia, whether cancer cell growth
is inhibited.
Inhibition of cancer cell growth may be evidenced, for example,
by arrest of cancer cells in a particular phase of the cell cycle,
e.g., arrest at the G2/M phase of the cell cycle. Inhibition of
cancer cell growth can also be evidenced by direct or indirect measurement
of cancer cell or tumor size. In human cancer patients, such measurements
generally are made using well known imaging methods such as magnetic
resonance imaging, computerized axial tomography and X-rays. Cancer
cell growth can also be determined indirectly, such as by determining
the levels of circulating carcinoembryonic antigen, prostate specific
antigen or other cancer-specific antigens that are correlated with
cancer cell growth. Inhibition of cancer growth is also generally
correlated with prolonged survival and/or increased health and well-being
of the subject.
As used herein, the term "induces apoptosis" refers to
the promotion of a form of programmed cell death characterized by
DNA fragmentation. Apoptosis can be determined by methods known
in the art. For example, kits are commercially available that detect
the presence of fragmented DNA by in situ immunohistochemistry (e.g.,
Apoptag, available from Intergen, Purchase, N.Y.). Additionally,
apoptosis can also be determined by FACS analysis, in which apoptotic
cells exhibit a sub-G1 DNA content, indicating DNA fragmentation.
As used herein, an "antibody" refers to a peptide, polypeptide,
or protein comprising one or more peptides or polypeptides substantially
or partially encoded by at least one immunoglobulin nucleic acid
molecule or immunoglobulin gene or fragment of at least one immunoglobulin
molecule or immunoglobulin gene. The recognized immunoglobulin genes
include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant
region genes, as well as myriad immunoglobulin variable region genes.
Light chains are classified as either kappa or lambda. Heavy chains
are classified as gamma, mu, alpha, delta, or epsilon, which in
turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively. A typical immunoglobulin (e.g., antibody) structural
unit comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light"
chain (about 25 kD) and one "heavy" chain (about 50 70
kD). The N-terminus of each chain defines a variable region of about
100 to 110 or more amino acids primarily responsible for antigen
recognition. The terms "variable light chain" (VL) and
"variable heavy chain" (VH) refer to these light and heavy
chains, respectively. Antibodies exist as intact immunoglobulins
or as a number of well characterized fragments produced by digestion
with various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce F(ab)'2,
a dimer of Fab which itself is a light chain joined to VH-CH1 by
a disulfide bond. The F(ab)'2 may be reduced under mild conditions
to break the disulfide linkage in the hinge region, thereby converting
the (Fab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially
a Fab with part of the hinge region (see Fundamental Immunology,
W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description
of other antibody fragments). While various antibody fragments are
defined in terms of the digestion of an intact antibody, one of
ordinary skill in the art will appreciate that such Fab' fragments
may be synthesized de novo either chemically or by utilizing recombinant
DNA methodology. Thus, the term "antibody", as used herein,
also includes antibody fragments either produced by the modification
of whole antibodies or synthesized de novo using recombinant DNA
methodologies. Antibodies include single chain antibodies, including
single chain Fv (sFv) antibodies in which a VH and a VL are joined
together (directly or through a peptide linker) to form a continuous
polypeptide. One preferred antibody is a single-chain antibody derived
from a human germline.
An "antigen-binding fragment" of an antibody is a peptide
or polypeptide fragment of the antibody that binds an antigen. An
antigen-binding site is formed by those amino acids of the antibody
that contribute to, are involved in, or affect the binding of the
antigen. See Scott, T. A. and Mercer, E. I., CONCISE ENCYCLOPEDIA:
BIOCHEMISTRY AND MOLECULAR BIOLOGY (de Gruyter, 3d ed. 1997) and
Watson, J. D. et al., RECOMBINANT DNA (2d ed. 1992), each of which
is incorporated herein by reference in its entirety for all purposes.
The term "antibody fragment" also includes any synthetic
or genetically engineered protein that acts like an antibody by
binding to a specific antigen to form a complex.
The terms "active agent", "drug" and "pharmacologically
active agent" are used interchangeably herein to refer to a
chemical material or compound which, when administered to a subject
induces a desired pharmacologic effect, and is intended to include
a diagnostic or therapeutic agent, including radionuclides, drugs,
anti-cancer agents, toxins and the like. Preferably, the term active
agent includes proteins, glycoproteins, natural and synthetic peptides,
alkaloids, polysaccharides, nucleic acid molecules, small molecules
and the like. More preferably, the term active agent refers to proteins.
An exemplary active agent is urease.
A "pH-sensitive" active agent refers to an active agent
whose ability to induce a desired pharmacologic effect depends,
at least in part, on the pH of the surrounding extracellular environment.
The term "clearing agent", as used herein, refers to
an agent capable of binding, complexing or otherwise associating
with an administered moiety, e.g., targeting moiety-ligand, targeting
moiety-anti-ligand or anti-ligand alone, present in the recipient's
circulation, thereby facilitating circulating moiety clearance from
the recipient's body, removal from blood circulation, or inactivation
thereof in circulation. The clearing agent is preferably characterized
by physical properties, such as size, charge, configuration or a
combination thereof, that limit clearing agent access to the population
of target cells recognized by a targeting moiety used in the same
treatment protocol as the clearing agent.
The term "imaging agent" is meant to refer to compounds
which can be detected.
The term "adjuvant" refers to a substance or agent added
to a formulation or composition to aid the operation of the main
ingredient.
The terms "interstitial" and "extracellular"
fluid refer to the fluid lying between or bathing the cells of mammals.
The terms "subject", "individual" and "patient"
are used interchangeably herein to refer to any target of the treatment.
Also provided by the present invention is a method of treating tumor
cells in situ, or in their normal position or location, for example,
neoplastic cells of breast or prostate tumors. These in situ tumors
can be located within or on a wide variety of hosts; for example,
human hosts, canine hosts, feline hosts, equine hosts, bovine hosts,
porcine hosts, and the like. Any host in which is found a tumor
or tumor cells can be treated and is in accordance with the present
invention. A subject thus includes a vertebrate, preferably a mammal,
more preferably a human.
By "target cell retention time" is intended the amount
of time that a urease molecule or other active agent remains at
the target cell surface or within the target cell.
As used herein, the term "conjugate" encompasses chemical
conjugates (covalently or non-covalently bound), fusion proteins
and the like.
The terms "protein", "polypeptide" or "peptide",
as used herein, refer interchangeably to a biopolymer composed of
amino acid or amino acid analog subunits, typically some or all
of the 20 common L-amino acids found in biological proteins, linked
by peptide intersubunit linkages, or other intersubunit linkages.
The protein has a primary structure represented by its subunit sequence,
and may have secondary helical or pleat structures, as well as overall
three-dimensional structure. Although "protein" commonly
refers to a relatively large polypeptide, e.g., containing 100 or
more amino acids, and "peptide" to smaller polypeptides,
the terms are used interchangeably herein. That is, the term "protein"
may refer to a larger polypeptide, as well as to a smaller peptide,
and vice versa.
A "modulator of urease" is either an inhibitor of urease
or an enhancer of urease.
An "inhibitor of urease" comprises a molecule or group
of molecules that interferes with: (1) the expression, modification,
regulation, activation or degradation of urease; or (2) one or more
of the normal functions of urease. The normal functions of urease
include the hydrolysis of urea, leading to the production of carbamate
and ammonia. An inhibitor "acts directly on urease" when
the inhibitor binds to urease via electrostatic or chemical interactions.
Such interactions may or may not be mediated by other molecules.
An inhibitor acts "indirectly on urease" when its most
immediate effect is on a molecule other than urease which influences
the expression, activation or functioning of urease.
An "enhancer of urease" comprises a molecule or group
of molecules that enhances: (1) the expression, modification, regulation
or activation of urease; or (2) one or more of the normal functions
of urease. An enhancer acts "indirectly on urease" when
its most immediate effect is on a molecule other than urease which
influences the expression, activation or functioning of urease.
The term "pharmaceutical composition" means a composition
suitable for pharmaceutical use in a subject, including an animal
or human. A pharmaceutical composition generally comprises an effective
amount of an active agent and a carrier, including, e.g., a pharmaceutically
acceptable carrier.
A "pharmaceutically acceptable formulation" comprises
a formulation that is suitable for administering the active agent
(e.g., urease or urease modulator) in a manner that gives the desired
results and does not also produce adverse side effects sufficient
to convince a physician that the potential harm to a patient is
greater than the potential benefit to that patient. The basic ingredient
for an injectable formulation is typically a water vehicle. Aqueous
vehicles that are useful include sodium chloride (NaCl) solution,
Ringer's solution, NaCl/dextrose solution, and the like. Water-miscible
vehicles are also useful to effect full solubility of the active
agent. Antimicrobial agents, buffers and antioxidants may be useful,
depending on the need. Similarly, a "pharmaceutically acceptable"
salt or a "pharmaceutically acceptable" derivative of
a compound, as provided herein, is a salt or other derivative which
is not biologically or otherwise undesirable.
A "therapeutic treatment" is a treatment administered
to a subject who displays symptoms or signs of pathology, disease,
or disorder, in which treatment is administered to the subject for
the purpose of diminishing or eliminating those signs or symptoms
of pathology, disease, or disorder. A "therapeutic activity"
is an activity of an agent, such as a nucleic acid, vector, gene,
polypeptide, protein, substance, or composition thereof, that eliminates
or diminishes signs or symptoms of pathology, disease or disorder,
when administered to a subject suffering from such signs or symptoms.
A "therapeutically useful" agent or compound (e.g., nucleic
acid or polypeptide) indicates that an agent or compound is useful
in diminishing, treating, or eliminating such signs or symptoms
of a pathology, disease or disorder.
The term "small molecule" includes a compound or molecular
complex, either synthetic, naturally derived, or partially synthetic,
and which preferably has a molecular weight of less than 5,000 Daltons.
More preferably, a small molecule has a molecular weight of between
100 and 1,500 Daltons.
As used herein, "effective amount" or "pharmaceutically
effective amount" of an active agent refers to an amount sufficient
to derive a measurable change in a physiological parameter of the
target cell or subject and/or to provide or modulate active agent
expression or activity through administration of one or more of
the pharmaceutical dosage units. Such effective amount may vary
from person to person depending on their condition, height, weight,
age, and/or health, the mode of administering the active agent (e.g.,
urease or urease modulator), the particular active agent administered,
and other factors. As a result, it may be useful to empirically
determine an effective amount for a particular patient under a particular
set of circumstances.
The term "specific therapeutic efficacy" of an active
agent as used herein means the amount of an active agent which is
required to achieve a specified amount or degree of therapeutic
efficacy, e.g., by inhibition of cancer cell growth or killing of
cancer cells or shrinkage of tumor size.
All publications and patents cited herein are expressly incorporated
herein by reference for the purpose of describing and disclosing
compositions and methodologies which might be used in connection
with the invention.
II. Compositions Used in the Treatment Methods
This section considers the urease composition and anti-cancer compounds
used in practicing the method of the invention.
A. Urease Composition
As noted above, the urease composition includes urease enzyme and
may additional include a targeting agent associated with the enzyme
for localizing urease at a target, e.g., tumor site. The urease
may be of any origin, including, e.g., bacteria, plants, fungi and
viruses. A number of studies have provided detailed information
about the genetics of ureases from a variety of evolutionarily diverse
bacteria, plants, fungi and viruses (Mobley, H. L. T. et al. (1995)
Microbiol. Rev. 59: 451 480; Eur J. Biochem., 175, 151 165 (1988);
Labigne, A. (1990) International publication No. WO 90/04030; Clayton,
C. L. et al. (1990) Nucleic Acid Res. 18, 362; and U.S. Pat. Nos.
6,248,330 and 5,298,399, each of which is incorporated herein by
reference). Of particular interest is urease that is found in plants
(Sirko, A. and Brodzik, R. (2000) Acta Biochim Pol 47(4): 1189 95).
One exemplary plant urease is jack bean urease, which is described
in Examples 2 3. An exemplary amino acid sequence of jack bean urease
is represented by SEQ ID NO: 1 below.
Useful urease sequences may be identified in public databases,
e.g., Entrez (www.ncbi.nlm.nih.gov/Entrez). Additionally, primers
that are useful for amplifying ureases from a wide variety of organisms
may be utilized by employing the CODEHOP (COnsensus-DEgenerate Hybrid
Oligonucleotide Primer) as described in Rose, et al. (1998) Nucl.
Acids Res. 26:1628.
The urease may contact the tumor cells, be positioned in the extracellular
environment or interstitial fluid surrounding the tumor cells, or
be expressed within the cancer cells or cells nearby the cancer
cells. While not wishing to be bound by any specific molecular mechanisms
underlying the successful inhibition of growth of cancer cells by
urease, the urease compound acts to raise the pH of interstitial
fluid in which the cancer cells are bathed, by conversion of urea,
which is typically present in an amount between about 1 8 mM, typically
about 1.5 5 mM urea, in interstitial or extracellular fluid, to
ammonia and carbamate (FIGS. 1A 1D). The environment around a cancer
cell is typically acidic, e.g., about 6.8. (Webb, S. D., et al.
(2001) Novartis Found Symp 240:169 81). Thus, by raising the pH
of the extracellular environment in this manner, growth of the cancer
cell is inhibited. Accordingly, addition of the active agent in
certain embodiments of the invention causes the pH of the interstitial
fluid to be raised by about 0.1 pH unit, e.g., 0.1 0.5 pH units
or greater.
Thus, active agents of the urease composition include the naturally
occurring forms of urease as well as functionally active variants
thereof. The nature of these active variants is more fully described
in the above cited, co-owned U.S. patent application (publication
No. 2004/0115186 A1, also referred to herein as the '186 application),
which is incorporated herein by reference.
A1 Targeting Agent
The urease composition employed in the method of the invention
may include a chemical entity associated with urease to enhance
its localization at target cancer cells. A variety of associated
chemical entities are contemplated and are described in the above-cited,
co-owned '186 application. One preferred chemical entity is covalently
bound targeting agent, also as detailed in the '186 application.
As described there, targeting moieties useful in localizing the
urease composition to a target site include antibodies and antibody
fragments, peptides, and hormones. Proteins corresponding to known
cell surface receptors (including low density lipoproteins, transferrin
and insulin), fibrinolytic enzymes, anti-HER2, platelet binding
proteins such as annexins, and biological response modifiers (including
interleukin, interferon, erythropoietin and colony-stimulating factor)
are also contemplated targeting moieties.
Oligonucleotides, e.g., antisense oligonucleotides that are complementary
to a portion of a target cell nucleic acid, may be used as targeting
moieties in the present invention. Targeting moieties may also be
oligonucleotides that bind to a target cell surface. Analogs of
the above-listed targeting moieties that retain the ability to bind
to a defined target cell population may also be used as targeting
moieties.
Preferred targeting moieties of the present invention are antibodies,
peptides, oligonucleotides or the like, that are reactive with an
antigen on the surface of a target cell. Both polyclonal and monoclonal
antibodies which are either available commercially or described
in the literature may be employed. The antibodies may be whole antibodies
or fragments thereof. Monoclonal antibodies and fragments may be
produced in accordance with conventional techniques, such as hybridoma
synthesis, recombinant DNA techniques and protein synthesis. Useful
monoclonal antibodies and fragments may be derived from any species
(including humans) or may be formed as chimeric proteins which employ
sequences from more than one species. In one embodiment, the antibody
is a single-chain antibody derived form a mammalian germline, e.g.,
llama germline. Methods for preparing conjugate composition between
urease and such targeting agents are detailed in the above-cited
'186 application.
A2. Entrapped Active Agents
In certain embodiments, the invention contemplates the use of vesicles
such as liposomes and/or nanocapsules as chemical entities for the
delivery of an active agent or active agents, e.g., urease to cancer
cells. Such formulations may be preferred for the introduction of
pharmaceutically-acceptable formulations of the polypeptides, pharmaceuticals,
and/or antibodies disclosed herein. The formation and use of liposomes
is generally known to those of skill in the art. (See, e.g., Backer,
M. V., et al. (2002) Bioconjug Chem 13(3):462 7). In a preferred
embodiment, the disclosed composition may be entrapped in a liposome.
Particle-entrapped urease compositions suitable for use in the invention
are detailed in the above-cited '186 application.
A3. Urease Inhibitor or Activator
Active agent modulators are also contemplated as associated chemical
entities by the instant invention. A preferred active agent modulator
is a urease modulator. A "urease modulator" is either
an inhibitor of urease or an enhancer of urease. The modulator in
the compositions (e.g., pharmaceutical compositions) accordingly
may be selected from among all or portions of urease polynucleotide
sequences, urease antisense molecules, urease polypeptides, protein,
peptide, or organic modulators of urease bioactivity, such as inhibitors,
antagonists (including antibodies) or agonists. Preferably, the
modulator is active in treating a medical condition that is mediated
by, or ameliorated by, urease expression or urease activity.
An "inhibitor of urease" comprises a molecule or group
of molecules that interferes with: (1) the expression, modification,
regulation, activation or degradation of urease: or (2) one or more
of the normal functions of urease, including the hydrolysis of urea
leading to the production of carbamate and ammonia. An inhibitor
"acts directly on urease" when the inhibitor binds to
urease via electrostatic or chemical interactions. Such interactions
may or may not be mediated by other molecules. Where the inhibitor
acts directly on urease, such as where the inhibitor is a reversible
inhibitor, a preferred inhibitor will dissociate from urease when
urease is diluted in the blood circulatory system and/or when it
reaches its cellular target. An inhibitor acts "indirectly
on urease" when its most immediate effect is on a molecule
other than urease which influences the expression, activation or
functioning of urease.
Urease inhibitors, which serve to slow the conversion of urea to
ammonium ions, include but are not limited to hydroxamic acid derivatives
and other urea analogs, (e.g., acetohydroxamic acid), phosphoramide
derivatives (e.g., flurofamide), phosphates, thiols (e.g., 2-mercaptoethanol
etc.), boric acid, halogen compounds (e.g., fluorides etc.), and
cassia bark extract. Additional urease inhibitors are known to those
of skill in the art and are described in U.S. Pat. No. 4,824,783
(Apr. 25, 1989) which is incorporated herein by reference. The effect
of a urease inhibitor, such as acetohydroxamic acid (AHA), on urease
cytotoxicity in tumor cells in culture is seen in Example 9. In
this example, it is seen that tumor cells are protected from urease
cytotoxicity in the presence of a urease inhibitor. Therefore, possible
toxicity to the patient caused by local high concentrations of urease
can be mitigated through the use of a urease inhibitor, such as
AHA, especially when the enzyme is applied in bolus form. e.g.,
by IV drip, to reduce localized toxicity effects. AHA is commercially
available under the trade name Lithostat.RTM. and has been approved
for human use.
An "enhancer of urease" comprises a molecule or group
of molecules that enhances: (1) the expression, modification, regulation
or activation of urease; or (2) one or more of the normal functions
of urease. An enhancer "acts directly on urease" when
the enhancer binds to urease via electrostatic or chemical interactions.
Such interactions may or may not be mediated by other molecules.
An enhancer acts "indirectly on urease" when its most
immediate effect is on a molecule other than urease which influences
the expression, activation or functioning of urease.
In accordance with one aspect of the invention, a urease composition
includes a urease and an inhibitor, e.g., AHA, at a mole ratio of
urease:inhibitor sufficient to bind to all of the urease active
sites, e.g., all six active sites of a normal bacterial urease.
This composition therefore contains inhibitor and urease at a mole
ratio of 6:1 or greater, e.g., 10:1 inhibitor:urease. At the relatively
high concentration of urease employed injected or infused into a
patient, the majority of the inhibitor will be in bound form, thus
inhibiting strongly inhibiting the activity of the enzyme and reducing
enzyme toxicity at and near the site of administration. As the urease
is carried toward the target site, e.g., in the bloodstream, it
will become more dilute, shifting the kinetics of inhibitor binding
toward its unbound form, and thereby releasing the inhibitory effect
on the enzyme with migration toward the target.
A pharmaceutical composition that includes urease enzyme and a
urease inhibitor, such as the inhibitors previously described herein,
may include at least about 1000 Units urease, where the concentration
of enzyme and inhibitor will be determined by the solution volume
to be infused or injected. Thus, a composition injected in a total
volume of 10 ml will have a urease concentration 1/100 of that intended
slow IV infusion in a 1 liter volume. The concentration of urease
in the composition, when in aqueous form, is preferably at least
about 20 Units/ml.
B. Weakly Basic Anti-Cancer Agents
The method of the invention is based on the discovery herein that
exposing cancer cells to urease, i.e., by localizing urease at the
site of cancer in a mammalian subject, is effective in potentiating
the anti-cancer effect of a weakly basic anti-cancer compound that
is effective against that cancer. This effect may be exploited,
in accordance with the invention, to achieve a comparable therapeutic
effect at a reduced compound dose, e.g., a dose that is 2 5 fold
lower than that otherwise required, and/or to achieve a superior
therapeutic effect at the same or reduced dose.
Weakly basic anti-cancer compounds include any anti-cancer compound
having one or more ionizable primary or secondary amine groups,
and whose uptake into cancer cells may be affected by the intracellular/lower
extracellular pH gradient in the extracellular environment of the
cells. Exemplary weakly basic anti-cancer compounds include doxorubicin,
daunorubicin, mitoxantrone, epirubicin, mitomycin, bleomycin, vinca
alkaloids, such as vinblastine and vincristine, alkylating agents,
such as cyclophosphamide and mechlorethamine hydrochloride, antineoplastic
purine derivatives, including cladribine, fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine or tiazofurin, and anti-neoplastic pyrimidine
derivatives, including ancitabine, azacitidine, 6-azauridine, capecitabine,
carmofur, cytarabine, decitabine, doxifluridine, emitefur, enocitabine,
floxuridine, gemcitabine or tegafur.
Although the invention is directed potentiating the anti-cancer
effect of weakly basic anti-cancer drugs, urease also appears to
potentiate the anti-cancer effect of other anti-cancer drugs, such
as fluorouracil (see examples below). The potentiating effect may
be related to a shift in extracellular or intracellular pH caused
by urease, or may involve one or more other mechanisms related to
the anti-cancer effects of urease alone, detailed in the above-cited
'186 application. Thus, in a more general aspect, the present invention
contemplates potentiating the anti-cancer activity of any anti-cancer
compound, such as fluorouracil, whose activity is enhanced by the
presence, in the extracellular fluid of target cancer cells, of
an amount of urease effective to raise the pH of such extracellular
fluid.
C. pH-Sensitive Imaging Agents
Imaging agents may be included in the composition or in additional
compositions, as detailed in the above-cited '168 application. Of
particular interest for the present invention are pH sensitive imaging
agents that can be used to detect and monitor changes in the local
pH environment of cancer cells, e.g., the extracellular matrix of
a solid tumor. Thus, for example, in practicing the present invention,
it may be advantageous, in determining a suitable initial dose of
the urease composition, to monitor the subject tumor site for urease-induced
pH changes. Typically, an increase in pH of at least 0.1 pH units
is desired, with changes of between 0.1 to 0.5 pH units or greater
being typical.
Both luminescent cyclen-based lanthanide chelates and those primarily
yielding magnetic resonance signatures have been shown to be sensitive
to changes in pH. Luminescent probes used for sensing pH changes
typically detect changes in the fluorescence lifetime of the lanthanide
ion as a function of pH. Analogously, magnetic resonance contrast
agents which modulate the water proton relaxivity via changes in
pH are useful in the instant invention. In both cases, by changing
the pH in a given system, one can envision agents with enhanced
contrast.
Accordingly, a pH sensitive contrast agent is utilized at or near
the cancer cell. The cancer cell or cells are also exposed to a
urease composition containing urease enzyme to cause a change in
pH at or near the cancer cell. In this way, a change in pH causes
the nuclear magnetic resonance relaxation properties of water protons
or other nuclei in the aqueous medium to be changed in a manner
that is reflective of pH. Examples of pH sensitive contrast agents
that may be utilized include those agents that contain a lanthanide
metal, such as Ce, Pr, Nd, Sm, Eu, Gd, Db, Dy, Ho, Er, Tm, Yb, and
the like, or another paramagnetic element, such as Fe, Mn, or the
like. Specific contrast agents that may be utilized include GdDOTA4AmP(5-)
which is described in Magn Reson Med. 2003 February;49(2):249 57,
and Fe(III)meso-tetra(4-sulfonatophenyl)porphine (Fe-TPPS4) as described
in Helpern et al. (1987) Magnetic Resonance in Medicine 5:302 305
and U.S. Pat. No. 6,307,372, which is incorporated herein by reference.
In addition, Gd based with polyion, as described in Mikawa et al.
Acad. Radiol (2002) 9(suppl 1):S109 S1111, may be used in the invention.
As another alternative, a shift reagent may be provided in the
aqueous medium surrounding the cancer cell. The shift reagent is
configured such that a change in pH affects the chemical shift properties
of the water protons or other nuclei in a manner that is reflective
of pH. The change in chemical shift properties may then be measured
using nuclear magnetic resonance to determine whether the active
agent is biologically active. Examplary shift reagents that may
be used include those containing a lanthanide metal, such as Ce,
Pr, Nd, Sm, Eu, Gd, Db, Dy, Ho, Er, Tm, or Yb, or another paramagnetic
element. Examples of specific shift reagents that may be utilized
include Tm(DOTP) (5-), the thulium (III) complex of 1,4,7,10-tetraazacylododecane-N,N',N'',N'''-tetra(methylenephospate).
Dy(PPP) (2)(7)-dysprosium tripolyphosphate, and the like.
In one embodiment of the invention, a dual-contrast-agent strategy
using two gadolinium agents, such as the pH-insensitive GdDOTP(5-)
and the pH-sensitive GdDOTA-4AmP(5-), may be utilized to generate
pH maps by MRI, as described in Magn Reson Med (2003) February;
49(2):249 57. Preferred agents for use with PET scan include 13N
and fluorodeoxyglucose (FDG).
III. Method of Potentiating a Weakly Basic Anticancer Compound
The present invention provides a method of potentiating the specific
therapeutic activity of a weakly basic anti-cancer compound in the
treatment of a mammalian cancer that is responsive to that compound.
In one general embodiment, a method includes administering to the
subject an amount of a urease composition which, when given in the
presence of urease, e.g., either by co-administration of urease
and the compound or following administration or urease, is effective
to reduce the amount of the compound needed to achieve a selected
anti-cancer effect, e.g., tumor shrinkage.
The degree of potentiation achievable with the invention will depend
upon a number of factors that can be readily assessed during treatment,
and the compound dosaging can be adjusted accordingly during treatment.
To the extent the potentiation of weakly basic anti-neoplastic agents
is attributable to a shift in the external/internal pH gradient
of the cancer cells, the extent of potentiation will depend on the
extent to which the shift in gradient enhances drug uptake. For
example, compounds that are readily taken up with ambient gradients
may show less potentiation than those whose uptake is strongly affected
by a pH change in the range 6.8 to about 7.2 or higher. Similarly,
where the cancer cells have developed drug resistance that relies
on pumping drug out of the cells, the urease potentiation effect
may be less pronounced. That is, the degree or extent of potentiation
achieved will depend on a variety of factors, including the pK or
pK.sub.as of the drug amine group(s); the presence or absence of
drug-resistance mechanisms in the cancer cells, including those
related to drug transport; the presence or absence of drug-specific
transport mechanisms in the cancer cells; and the solubility of
the drug in a lipid membrane. In a typical method, the extent of
potentiation is between 2 and 5 fold; that is, the amount of compound
needed to produce a given anti-cancer effect is 2- to 5-fold lower
when the cells are pretreated by urease.
In selecting a weakly basic anti-cancer drug for treating a particular
cancer, the physician will typically select a compound that has
been routinely used or has demonstrated effectiveness against the
particular type of cancer being treated. Table 2 below provides
a partial list of weakly basic anti-cancer drugs that have been
shown to be effective against a variety of human cancer types, as
indicated in the table.
TABLE-US-00001 TABLE 2 Weakly Basic Agents Used in Cancer Treatment
Which Can be Potentiated by Increased Extracellular pH Compound
Application Comments Doxorubicin (Adriamycin .RTM., Breast Cancer
An anthracycline Rubex .RTM., Doxil .RTM., Caelyx .RTM., Ovarian
Cancer antibiotic from the fungus Myocet .RTM.) Lung Cancer Strep.
It interferes with Bladder Cancer DNA production in the cell Gastric
Cancer Thyroid Cancer Daunomycin Acute Myelogenous An anthracycline
(Cerubidine .RTM.) Leukemia antibiotic. It interferes Acute Lymphocytic
with DNA production in Leukemia the cell Epirubicin (Ellence .RTM.)
Breast Cancer An anthracycline antibiotic. It interferes with DNA
production in the cell Vinblastine (Velban .RTM., Breast Cancer
A vinca alkaloid. It Velbe .RTM.) Hodgkin's Disease inhibits cell
division during Kaposi's Sarcoma early mitosis Testicular Cancer
Vincristine (Oncovin .RTM., Acute Leukemia A vinca alkaloid. It
Vincasar PFS .RTM., Vincrex .RTM.) Rhabdoyosarcoma inhibits cell
division during Neuroblastoma early mitosis Lymphorecticular Neoplasms
Mitoxantrone Prostate Cancer An intercalating agent (Novantrone
.RTM.) Acute Nonlymphocytic that interacts with DNA Leukemia and
blocks DNA synthesis Bleomycin (Blenoxane .RTM.) Squamous Cell A
mixture of cytotoxic Carcinomas antibiotics, thought to Hodgkin's
Disease inhibit DNA synthesis Non-Hodgkin's Lymphoma and, to a lesser
degree, Testicular Cancer RNA and protein synthesis Mitomycin (Mutamycin
.RTM.) Gastric Cancer An alkylating agent. It is Anal Cancer thought
to inhibit cell Colon Cancer growth by causing Breast Cancer inter/intrastrand
Non-Small Cell Lung crosslinkages in DNA, Cancer thereby causing
Head and Neck Cancer miscoding, breakage and Small Bladder Papillomas
replication failures Pancreatic Cancer Cervical Cancer Mechlorethamine
Hodgkin's Disease An alkylating agent. It is Hydrochloride thought
to inhibit cell (Mustargon .RTM.) growth by causing inter/intra
strand crosslinkages in DNA, thereby causing miscoding, breakage
and replication failures
In the treatment method, the subject is typically pretreated with
a selected dose of the urease composition. Where a urease composition
is injected directly into a tumor, an exemplary dose is 0.1 to 1,000
international units urease activity per mm.sup.3 tumor. For example,
and assuming a relatively uniform distribution of the urease in
the tumor is achieved, a dose of between 0.5 and 5 international
units may be suitable. The placement of the injection needle may
be guided by conventional image guidance techniques, e.g., fluoroscopy,
so that the physician can view the position of the needle with respect
to the target tissue. Such guidance tools can include ultrasound,
fluoroscopy, CT, MRI, pr PET scan.
The effectiveness or distribution of the administered urease dose
may be monitored, during or after direct injection of urease into
the tumor, by monitoring the tumor tissue by a tool capable of detecting
changes in pH within the cancerous tissue region of the subject.
Such tools may include a pH probe that can be inserted directly
into the tumor, or a visualization tool, such as magnetic resonance
imaging (MRI), computerized tomography (CT), or fluoroscopy. MRI
interrogation may be carried out in the absence of additional imaging
agents, based simply on differences in magnetic properties of tissue
as a function of pH. CT or fluoroscopic imaging may require an additional
pH-sensitive imaging agent whose opacity is affected by the pH of
the tissue medium. Such agents are well known to those of skill
in the art.
Before any urease injection, the tumor tissue can be visualized
by its lower pH relative to surrounding normal tissue. Thus, the
normal tissue may have a normal pH of about 7.2, whereas the tumor
tissue may be 0.1 to 0.4 or more pH units lower. That is, before
any urease is injected, the extent of tumor tissue can be defined
by its lower pH. Following urease administration, the pH of the
tumor region having urease will begin to rise, and can be identified
by comparing the resulting images with the earlier pre-dosing images.
By interrogating the tissue in this manner, the degree of change
in pH and extent of tissue affected may be monitored. Based on this
interrogation, the physician may administer additional urease composition
to the site, and/or may administer composition at additional areas
within the tumor site. This procedure may be repeated until a desired
degree of pH changes, e.g., 0.2 to 0.4 pH units, has been achieved
over the entire region of solid tumor.
Where the urease is administered parenterally by a method other
than direct injection, e.g., by IV drip, an exemplary dose of the
urease is 100 100,000 international units urease activity/kg subject
body weight, or alternatively, between about 25 2000 pmoles, preferably
25 500 pmolesurease/kg subject body weight. As noted herein, the
urease composition in this method preferably includes a targeting
agent for targeting urease to the cancer cells, e.g., site of solid
tumor, or for sequestering urease, e.g., in liposomal form, selectively
at the tumor site. The composition described above containing urease
in combination with stoichiometric amounts of an inhibitor is advantageous
in intravenous administration for the reasons discussed above. As
above, imaging techniques that are sensitive to changes in tissue
pH, may be used to monitor urease dosing.
Where the urease composition is given by IV injection or drip,
the urease is typically allowed to clear from the body for a period
of at least 24 hours prior to administration of the anti-cancer
compound. The rate of clearance can be accelerated, if desired,
by administering a clearing agent, such as an anti-urease antibody,
that facilitates clearance by non-renal clearance mechanisms. Such
methods are well known.
Once an adequate dose of the urease composition has been administered
and localized at the target site, and preferably after a period
that allows for clearance of a significant portion of the circulating
urease, the anti-cancer compound is administered by conventional
dosing methods, e.g., oral administration or IV drip. According
to an important feature of the invention, the urease localized at
the cancer site potentiates the anti-cancer drug, allowing a significantly
reduced dose of the compound to be administered, and/or a greater
therapeutic efficacy to be achieved at the same or reduced dose.
As noted above, the reduction in dose of anti-cancer compound is
typically 2- to 5-fold over that in the absence of urease, and these
values can be used as a guide to dosing in the method. Thus, the
physician may start with a dose that is 1/2 that of conventional
dosing, and if a good therapeutic result is achieved, the dose could
be reduced further until a lowest effective dose is reached. Preferably,
the dose administered will be the highest dose compatible with patient
comfort and lack of undesired side effects, such as nausea, alopecia,
malaise, and loss of a significant portion of white blood cells,
allowing effective cancer therapy with substantially reduced discomfort
and long-term risk to the patient.
In a typical anti-cancer treatment, the anti-cancer drug is administered
at twice or three-times weekly dosing over a 2 4 week period, followed
by a recovery and observation period of one to several months. In
the present invention, this regimen may be supplemented by urease
administration preceding each dosing, or preceding each dosing period,
e.g., once a week or once every other week during the period of
compound treatment. The frequency of urease administration may be
assessed during treatment by periodic assessment of localized pH
levels at the cancer site, using the above pH monitoring tools.
During the course of treatment, the size and shape of a tumor may
be monitored by diagnostic tools, such as those described above.
In particular, the subject can be interrogated with a diagnostic
tool capable of detecting changes in extracellular pH in a subject's
tissue, as described above. The diagnostic tool is preferably a
pH-sensitive diagnostic agent, such as an imaging, contrast or shift
reagent, as described in Section II, above, capable of localizing
in the tumor that may be administered prior to, following or concurrently
with the active agent. A tissue region is identified within the
subject that shows an elevation in extracellular pH following the
administration. Any tool capable of identifying the diagnostic agent
may be used to detect the agent, such as MRI, PET scan, and the
like, as described above.
In one embodiment, the method includes administering urease to
the subject employed in an anti-cancer therapy, and the identification
is used for detecting the localization of urease in a solid tumor.
The identifying may be used for monitoring the change in size and
shape of the tumor in response to urease administration.
In one embodiment employing PET scan, the subject is administered
13N-labelled ammonia. The patient is then administered urease in
an amount effective to reach the tumor site. The urease hydrolyzes
urea to produce non-labelled ammonia. Over time, the labelled ammonia
is diluted or displaced, causing a gradual clearing on the scan.
In another embodiment employing PET scan, the subject is administered
13N-labelled urea. The patient is then administered urease in an
amount effective to reach the tumor site. The urease hydrolyzes
the labelled urea to produce labelled ammonia, which could be detected
on the scan.
From the foregoing, it can be seen how various objects and features
of the invention are met. The invention provides an effective and
relatively simple method for potentiating the therapeutic effect
of a large class of anti-cancer compounds. This potentiation allows
a comparable or superior anti-cancer effect to be achieved at a
lower dose of compound, thus reducing patient discomfort, health
risks to the patient, and improving patient compliance and prospects
for recovery. The dosing and localization of urease can be readily
monitored, according to localized pH changes, and these same changes
can be used to monitor the efficacy of the treatment method, and
to monitor the urease dosing administered.
The following examples further illustrate the invention described
herein and are in no way intended to limit the scope of the invention.
A. EXAMPLE 1
A1. Peptide Synthesis
Peptides were prepared by solid-phase synthesis methodology using
conventional N-t-butyloxycarbonyl (t-Boc) chemistry. Peptides were
cleaved from the resin by reaction with hydrogen fluoride (20 ml/g
resin) containing 10% anisole and 2% 1,2-ethanedithiol for 1.5 h
at 4.degree. C. Crude peptides were washed with cold ether, and
extracted from the resin with glacial acetic acid and freeze-dried.
Synthetic peptide was purified by reversed-phase HPLC on a Zorbax
semi-preparative C-8 column (250.times.10 mm I.D., 6.5-.mu.m particle
size, 300-.ANG. pore size) with a linear AB gradient (ranging from
0.2 to 1.0% B/min) at a flow rate of 2 ml/min, where solvent A is
aqueous 0.05% trifluoroacetic acid (TFA) and solvent B is 0.05%
TFA in acetonitrile. Homogeneity of the purified peptides was verified
by analytical reversed phased-HPLC, amino acid analysis and MALDI
mass spectrometry.
A2. Affinity Purification of Urease
The affinity column was prepared by reacting hydroxyurea to epoxy-activated
Sepharose 6B (Amersham Biosciences). Remaining active groups were
blocked using 1 M ethanolamine.
Purification was performed as follows. The column was equilibrated
with PEB (0.02 M phosphate, 1 mM EDTA, 1 mM .beta.-mercaptoethanol,
pH 7.0). A crude urease sample (FIG. 2) was applied (0.5 mg/ml in
PEB, total 8 ml). The column was washed with 15 ml of PB (0.02 M
phosphate, 1 mM .beta.-mercaptoethanol, pH 7.0). The column was
then washed with 8 ml of each of the following: PB+0.1 M NaCl, PB+0.5
M NaCl, and PB+0.95 M NaCl. The urease was eluted with 8 ml of EB
(0.2 M phosphate, 1 mM .beta.-mercaptoethanol, pH 4.6), collecting
1 ml fractions. Fractions were checked by reading OD at 280 nm (FIG.
3) and HPLC (C5 column) analysis. The column was stored in 0.01%
NaN.sub.3.
B. EXAMPLE 2
Preparation of the Urease-Coil Conjugate
Urease coil conjugate was prepared by dissolving 10 mg of Jack
bean Urease in 300 .mu.l of 2 mM phosphate buffer pH 7.2. Then 5
mg of the bifunctional cross-linker Sulfo-MBS was added to the solution
and the mixture was slowly stirred for one hour at room temperature.
The mixture was then dialyzed against 2 mM phosphate buffer at pH
7.2 to remove excess linker.
K-coil or E-coil with a C-terminal cys linker (1.5 mg) was added
to the linker-modified urease solution and slowly mixed for 3 hours
at room temperature. The coil urease conjugate was dialyzed against
fresh 2 mM phosphate buffer at pH 7.2 overnight to remove unconjugated
coil peptide. Dialyzed urease conjugate was lyophilized, then dissolved
in 1 mL of 2 mM phosphate buffer pH 7.2 and applied to sephadex
G75 column for further purification. The void volume fractions,
which contained the coil urease conjugate, were pooled, freeze-dried
and stored at 4.degree. C.
The purity of the conjugate and the ratio of the coil to urease
in the preparation were determined by amino acid analysis and MALDI
mass spectrometry using standard procedures.
C. EXAMPLE 3
Activity Assay of Urease and Urease Conjugate
The enzymatic activity of urease or urease conjugate was carried
out in a coupled enzyme reaction with glutamate dehydrogenase (GLDH).
The amount of NADH oxidized was determined by measuring the change
in absorbance at 340 nm (Kaltwasser, H. and Schlegel, H. G., Anal.
Biochem., 16, 132, 1966). The reagents used were: 0.10 M Potassium
phosphate buffer, pH 7.6; 1.80 M Urea prepared in phosphate buffer;
0.025 M Adenosine-5'-diphosphate (ADP) (10.7 mg/ml) in buffer; 0.008
M NADH (5 mg/ml) in phosphate buffer; 0.025 M .alpha.-Ketoglutarate
(3.7 mg/ml) in phosphate buffer; Glutamate dehydrogenase (GLDH)
solution, free from ammonium ions; 50 U/ml phosphate buffer prepared
fresh prior to assay. Urease solution was prepared by dissolving
in phosphate buffer to yield a concentration of 0.1 0.5 U/ml. This
solution was prepared fresh prior to assay.
Assay was initiated by adding the following 2.0 mL of Phosphate
buffer 2.40 ml, 0.10 ml each of urea, ADP, NADH, GLDH and .alpha.-Ketoglutarate
in a cuvette. The spectrophotometer was adjusted to 340 nm and 25.degree.
C. The cuvette with the added ingredients was placed in the spectrophotometer
at 25.degree. C. for 5 minutes to attain temperature equilibration
and then establish blank rate, if any, at 340 nm.
To initiate the enzymatic reaction 0.1 ml of the urease solution
was added to the cuvette. The changes in the absorbance at 340 nm
were recorded for 15 min. Enzyme activity was correlated with a
decrease in absorbance at 340 nm per min.
D. EXAMPLE 4
Preparation of Coil Antibody Conjugate
Materials include: (1) Rat Anti-hEGFR IgG2a (Serotec), 200 .mu.g/0.2
ml (i.e. 1 mg/ml); (2) E-coil (N-linker); (3) Sodium m-periodate
(Pierce); and (4) Bifunctional crosslinker, KMUH (Pierce).
Functional modification of E-coil was performed by performing the
following steps: a. Dissolve KMUH in DMSO to prepare a 10 mg/ml
solution (2.5 mg in 250 .mu.l of DMSO). b. Dissolve E-coil in PB
(.about.2 mg in 392 .mu.l of 10 mM PB, pH 7.4+4 .mu.l of TCEP, 100
mM stock) c. Add 1 .mu.l of Tris (2 M) to neutralize the E-coil
solution d. Add E-coil solution to the KMUH solution and incubate
at R.T. for 2 hr e. Keep solution at 4.degree. C. overnight f. Next
morning, centrifuge at 12000 rpm for 5 min. to remove insoluble
precipitate. g. Remove KMUH and DMSO on a C8 HPLC column (0 20%
acetonitrile/H.sub.2O with 0.05% TFA) and collect all peptide fractions
(75% acetonitrile). g. Lyophilize the peptide fractions and check
by MS.
The antibody was oxidized by the following steps: a. For each 2
mg of antibody, weigh 20 mg of periodate in an amber vial. b. Add
2 ml of PBS, pH 7.2 and 2 ml of stock antibody to the vial (final
[antibody] is 0.5 mg/ml) and gently swirl until the periodate powder
was dissolved. c. Incubate at room temp. for 30 min. d. Remove periodate
by dialyzing 3 times vs 100 mM acetate buffer, pH 5.5.
Conjugation was performed by the following steps: a. Concentrate
oxidized antibody (.about.2 mg in 4 ml) using Millipore Ultrafree
Filter units (30 k MWc/o). b. Add 75 .mu.l of the functionalized
E-coil solution (4 .mu.g/.mu.l ddH.sub.2O) to half of the oxidized
antibody solution (containing .about.0.75 mg of antibody in acetate
buffer, pH 5.5). c. Incubate at room temp. for 2 hr with shaking.
d. Purify the antibody mixture using a Protein G column (See FIG.
4). e. Compare and analysis of sample (before and after affinity
purification).
E. EXAMPLE 5
Biacore Analysis of Coil Urease Conjugate and Coil Antibody Conjugate
Cysteine containing K-coil peptide or the E-coil peptide was covalent
coupled to the Pioneer B1 biosensor chip according to the manufacturer
suggested protocol. Briefly, the dextran surface of the sensor chip
was first activated with NHS/EDC (15 .mu.l) followed by addition
of PDEA (20 .mu.l). K-coil (or E-coil (50 .mu.g/ml) in 10 mM sodium
acetate buffer pH 4.3 was injected and allowed to react to give
a surface density of approximately 200 400 RU. Remaining activated
groups were then blocked by injection (10 .mu.l) of a 50 mM cysteine,
1 M NaCl, 0.1 M formate, pH 4.3 deactivation solution.
Kinetic experiments were performed on a BIAcore3000 instrument
at 25.degree. C. Each biosensor run consisted of (1) a 600 s sample
injection phase (coil urease or coil antibody), (2) a 600 s dissociation
phase, and (3) a 2.times.15 s regeneration phase (6M guanidine HCl).
A flow rate of 5 .mu.l/min was maintained throughout the cycle.
PBS was used as a buffer. The SPR signal was recorded in real time
with sampling at every 0.5 s and plotted as RU versus time (sensorgram).
Each sensorgram obtained was corrected for bulk refractive index
changes by subtracting the corresponding sample injection cycle
on a blank cell surface.
F. EXAMPLE 6
Animal Studies
Athymic nu/nu female mice with human mammary gland adenocarcinoma
xenografts were used for testing. Animals selected were generally
5 to 7 weeks of age, and their body weights at treatment commencement
range from approximately 15 to 28 grams.
MCF-cells were used to generate the xenografts. The cells were
grown in MEM media supplemented with Penicillin/Streptomycin 50
U/ml, L-glutamine 2 mM, Sodium pyruvate, nonessential amino acids,
vitamins, and 10% FBS; The cell incubator was maintained with 5%
CO.sub.2, 37.5.degree. C., and 80% humidity. The cells were harvested
with 0.25% (w/v) trypsin-0.03% (w/v) EDTA solution. Approximately
1.times.10.sup.6 cells in 100 .mu.L was injected subcutaneously
to the right flank of each mouse.
Tumor growth was allowed to proceed for about 6 8 days allowing
the size of the tumor to reach at least 2 4 mm in diameter. Doses
were administered via intratumor injection. The dose volume for
each animal was 50 mL. Each solid tumor was injected with the given
dose of test article in a "fanning fashion". Tumor volumes
were taken by external caliper measurements. Body weights were taken
at the start of the trial and at time of sacrifice.
Results, as shown in Table 3 below, show that tumors were not perceptible
24 hours following treatment.
TABLE-US-00002 TABLE 3 Successful Treatment of Tumors in Mice Mouse
1 2 3 4 5 6 MCF cell injected 0.8 .times. 10.sup.6 0.8 .times. 10.sup.6
0.8 .times. 10.sup.6 0.8 .times. 10.sup.6 1.3 .times. 10.sup.6 0.8
.times. 10.sup.6 Tumor size before 22.5 mm.sup.3 33.5 mm.sup.3 15.6
mm.sup.3 31.1 mm.sup.3 32.5 mm.sup.3 8.2 mm.sup.3 treatment Urease
amount 50 U/50 uL 50 U/50 uL 50 U/50 uL 50 U/50 uL 40 U/50 uL 10
U/50 uL injected Tumor size post not not Not Not Not Not injection
(24 hours) perceptible perceptible perceptible perceptible perceptible
percep- tible
G. EXAMPLE 7
Urease-Mediated Antitumor Effects in vitro
This example shows that urease is cytotoxic to A549 and MDA-MB-231
cells in culture.
Materials
Urease from Canavalia ensiformis (jack beans) was obtained from
BioVectra Ltd. (PEI, Canada) and further purified by acid precipitation
and ion exchange chromatography to remove the two major contaminants,
canavalin and concanavalin A. The purity of the enzyme was >96%
as determined by SDS polyacrylamide gel electrophoresis, HPLC and
mass spectrometry. One unit of urease is defined as the production
of 1 .mu.mole of ammonia per minute at 25.degree. C. and pH 7.6.
Urea, trypsin, phenazine ethosulfate (PES), sodium nitroprusside,
sodium hypochlorite solution, phenol, acetohydroxamic acid (AHA),
doxorubicin hydrochloride and vinblastine were purchased from Sigma
Chemical Co. (St. Louis, Mo.). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium (MTS) was purchased from Promega Corp. (Madison,
Wis.). Cell culture medium, serum, and antibiotics were obtained
from Invitrogen Life Technologies (ON, Canada). Female athymic nude
mice were supplied by Charles River Laboratories (Wilmington, Mass.).
Buffers used in the experiments: Dulbecco's phosphate-buffered saline
(D-PBS) and modified Krebs Ringer buffer (KRB) containing NaCl (98.3
mM), KCl (4.73 mM), KH.sub.2PO.sub.4 (1.19 mM), MgSO.sub.4 (1.19
mM), NaHCO.sub.3 (3.57 mM), D-Glucose (11.7 mM), Na.sub.2HPO.sub.4
(11.1 mM) and NaH.sub.2PO.sub.4 (2.77 mM) at pH 7.2 or 6.8.
Cell Culture and Viability Assay
Human breast cancer cell lines (MDA-MB-231 and MCF-7) and lung
cancer cell line (A549) were purchased from the American Type Culture
Collection (Manassas, Va.). Cells were grown in DMEM/F12 (MDA-MB-231
and A549) or MEM (MCF-7) containing 10% fetal bovine serum and 50
U/ml penicillin and 50 .mu.g/ml streptomycin at 37.degree. C. in
a humidified incubator with 5% CO.sub.2.
Colorimetric MTS assay was employed to determine cell viability.
MTS and PES were dissolved in D-PBS and filtered to prepare 2 and
1 mg/ml stock solutions, respectively. After the cells were treated
with test articles, the medium in the plate was replaced with 100
.mu.l/well of plain culture medium, followed by the addition of
20 .mu.l/well of MTS mix (MTS: PES at 20:1, vol/vol). The plate
was incubated for 1 2 hours at 37.degree. C. in a humidified, 5%
CO.sub.2 atmosphere. The absorbance of soluble formazan produced
by cellular reduction of MTS was measured at 490 nm with reference
at 630 nm using an ELx808IU Microplate Reader (Bio-tek Instruments
Inc., Winooski, Vt.).
In vitro Cytotoxicity of Urease
A549 cells (1.0.times.10.sup.5 cells/ml) or MDA-MB-231 cells (1.5.times.10.sup.5
cells/ml) were seeded into 96-well tissue culture plates (Becton
Dickinson Labware, N.J.) by transferring aliquot of 100 .mu.l of
the cell suspension to each well. The plate was incubated at 37.degree.
C. overnight. After incubation, the medium in the wells was removed
using a multichannel pipette. Urease and urea at various dilutions
were prepared in pre-warmed KRB, pH 7.2 and 50 .mu.l of each was
added to corresponding wells. After incubation for two hours at
37.degree. C., buffer from each replicate was pooled and subjected
to pH measurement and total ammonium determination as described
in Example 8. MTS cell viability assay was then performed on the
plate.
Results
Human tumor cell lines A549 and MDA-MB-231 were susceptible to
the enzymatic activity of jack bean urease. The survival rate of
both cell lines decreased as the cells were treated with 2 U/ml
of urease for 2 hours with an increasing amount of urea in the medium
(FIG. 6A). Urea alone (up to 40 mM as tested) was not toxic to the
cells during the 2-hour incubation period. In the presence of 2
U/ml of urease, the IC.sub.50 of urea were found to be 13 mM for
both cell lines (FIG. 6A). Furthermore, the cytotoxic effects were
also dependent on the availability of urease. With fixed urea concentration
at 20 mM in the medium, the IC.sub.50 of urease were found to be
0.22 and 0.45 U/ml, respectively, for A549 and MDA-MB231 cells (FIG.
6B). Sufficient amounts of ammonia produced by urease therefore
mediate cell death on both human lung and breast cell lines described
herein. Urease can therefore be used alone through the direct cytotoxicity
of ammonia.
It was found herein that significant amounts of canavalin and concanavalin
A contaminants were present in a commercial source of crystalline
urease as detected by HPLC and mass spectrometry. These contaminants
were able to increase in vitro cytotoxicity of the enzyme (unreported
observation).
H. EXAMPLE 8
Effect of Urease on pH and Ammonium Concentration of Culture Medium
of A549 Cells
This example shows that urease increases the total ammonium content
and the pH of culture medium from A549 cells in culture.
Methods
The total ammonium present in the incubation buffer obtained from
plates utilized in the in vitro cytotoxicity studies in Example
7 was determined by Berthelot's Indophenol reaction (Chaney, A.
L. and Marback, E. P. Clin. Chem. 8:130 132 (1962)). In brief, fresh
phenol solution (Solution A) was prepared by dissolving 165 mg of
phenol and 132 mg of NaOH pellets in 10 ml of water, and then 66
.mu.l of sodium nitroprusside solution (10 mg/ml) was added. Fresh
Solution B was prepared by adding 80 .mu.l of sodium hypochlorite
solution (10 13% chlorine) to 10 ml of water. Urease activity in
the pooled sample was quenched by adding 50 .mu.l of 1 N HCl to
100 .mu.l of sample. The acidified samples were then diluted 200
times and transferred to a 96-well microplate at 100 .mu.l/well,
followed by 50 .mu.l of Solution A and 50 .mu.l of Solution B. After
incubation at 37.degree. C. for 15 min, the plate was read at 630
nm using the microplate reader. The amount of ammonium ions present
in the sample was determined from the NH.sub.4Cl standard curve.
Cell culture and viability assays were performed as described in
Example 7.
Results
The antitumor effects of urease observed in Example 7 were exerted
through the hydrolysis of urea into ammonia (FIG. 6C) with a corresponding
elevation of pH (FIG. 6D) due to protonation of ammonia in aqueous
medium. FIGS. 6C and 6D were the total ammonium and pH measured
in the reaction buffer collected from experiments of FIG. 6A. It
was found that urease cytotoxicity (FIG. 6A) correlated well with
a corresponding increase of ammonium content and pH in the incubation
buffer (FIGS. 6C and 6D). The rise in pH of the control as shown
in FIG. 6D was probably due to autolysis of urea in aqueous medium
resulted in the generation of a total of 3 to 5 mM of ammonium.
However, separate experiments have shown that pH alone was insufficient
to cause any significant cytotoxicity on the two cell lines during
a two-hour incubation window (data not shown). Therefore, it was
the level of ammonium, or more specifically, availability of free
ammonia, that mediated the rapid cell killing effects. An augmented
pH in turn increases the availability of free ammonia to the cells
according to the following equation:
.times..times..times..times..times..times..times..times..degree..times..ti-
mes. ##EQU00001##
I. EXAMPLE 9
Effects of Acetohydroxamic Acid on Urease Cytotoxicity In Vitro
and the Ammonium Content of Culture Media
This example shows that acetohydroxamic acid decreases the total
ammonium content of cell culture medium and protects tumor cells
in the culture medium from urease cytotoxicity. Cells were cultured
and viability was determined as described in Example 7. The ammonium
content of culture medium was determined as described in Example
8.
Results
Acetohydroxamic acid (AHA) is a potent reversible inhibitor of
urease and is commercially available for the treatment of chronic
urea-splitting urinary infection. When AHA was added to the culture
buffer containing urease, it effectively inhibited the enzymatic
activity of urease (FIG. 7C) and restored the survival rate of A549
(FIG. 7A) and MDA-MB-231 (FIG. 7B) cells to normal level at dose
.gtoreq.2 mM. This result suggested that the cytotoxicity of urease
is solely due to its enzymatic activity. Similar results are shown
in FIG. 8A 8C at different levels of urea. Two millimolar AHA can
reduce ammonium production by DOS47 (urease) at 27 mM urea to a
level similar to that of 3 mM urea, and thus protects cells from
being killed (FIG. 7D). It is estimated that AHA at 0.8 mM can reduce
the ammonium production by half (FIG. 7D). AHA at 2.5 mM can almost
completely reverse the cytotoxic effects of DOS47 in KR-II buffer
containing 25 mM urea during a 2 hour incubation period for both
A549 and MB-231 cells (FIGS. 8E and 8F). AHA is not toxic to both
cell lines at concentrations as high as 12.5 mm (FIGS. 7E and F).
In comparing FIGS. 6 and 8, it can be seen that short term in vitro
urease treatment required much higher levels of urea than that of
longer term in vivo treatment.
J. EXAMPLE 10
Anti-Cancer Effects of Urease in vivo
This example shows that MCF-7 and A549 xenografts from mice are
susceptible to intratumoral injections of urease.
Methods
A549 xenografts: Female athymic nude mice (7 9 weeks old) were
injected subcutaneously in the right lateral thorax with 5.times.10.sup.6
human A549 lung cancer cells. When tumors reached 100 200 mg, the
tumor-bearing animals were randomly selected and sorted into four
groups. Group 1 contained ten untreated control mice. Group 2 contained
ten positive control mice treated with the reference chemotherapeutic
agent Cisplatin. Groups 3 and 4 contained 15 mice each that received
one of two different concentrations of urease (1 U or 4 U per injection).
Each animal received five doses of injection scheduled at 48 hours
between treatments (q2d.times.5). Urease was administered intratumorally,
whereas cisplatin was administered via an intravenous tail vein
injection.
Twenty-four hours after the fifth urease administration, five mice
from each group were sent for necropsy and tumor collection. In
addition, five mice from Groups 2 4 were euthanized and tumors were
collected, dissociated, and cultured for viability testing. The
remaining five animals from each group continued on study until
study termination. Tumor size was recorded using calibrated hand-held
Vernier calipers. Throughout the study, the length (L) and width
(W) of any tumors that developed were measured in millimeters. The
tumor weight in mg was calculated using the formula: (L.times.W2)/2.
Individual animal weights were taken twice weekly. The experimental
protocol was covered under Charles River Laboratories Institutional
Animal Care and Use Committee (IACUC).
MCF-7 xenograft: Female athymic nude mice were injected subcutaneously
with 1.8.times.10.sup.6 MCF-7 breast tumor cells. When tumor size
reached approximately 9 mg, eight mice were injected intratumorally
with two doses of urease (10 U/injection) at 48 hours interval (q2d).
Five mice were treated with saline as control. The condition of
all animals and tumor size were monitored 24 hours after inoculation
and then every other day. Animal care was in accordance with the
guidelines of the Canadian Council on Animal Care (CCAC). At the
end of the observation period, tumors were excised and prepared
for histology analyses.
Results
Both MCF-7 and A549 xenografts were susceptible to intratumoral
injections of urease. Growth of MCF-7 was completely stopped after
the second injection of 10 U of urease (FIG. 8A). Histological analysis
of tumors excised from the treated mice showed that the tumor mass
was dead and could not re-grow in culture medium.
In the case of A549 xenografts, delay in tumor growth was observed
in mice treated with 5 injections of low dose (1 U) or medium dose
(4 U) of urease (FIG. 8B). Growth regressions of the urease-treated
groups were similar to that of the positive control group treated
with 6 mg/kg Cisplatin (data not shown). Tumor tissue slices showed
significant necrotic area compared to the control. However, cell
viability was not affected (>82%) in dissociated tumors taken
from mice treated with urease. There were no significant differences
in body weight of the treatment and control groups in both xenograft
studies.
K. EXAMPLE 11
In vitro Anti-Cancer Effects of Urease in Combination with Selected
Anti-Neoplastic Drugs
This example shows that urease enhances the anti-cancer effects
of doxorubicin, vinblastine, fluorouracil, and mitoxantrone.
Methods
Combined Drug Assays
A549 and MDA-MB-231 plates were prepared as described in Example
7. Urease, urea and the 4 weak-base anticancer drugs, doxorubicin,
vinblastine, fluorouracil and mitoxantrone were prepared in pre-warmed
KRB, pH 6.8. After the medium in the plate was removed, 50 .mu.l
of urease, urea and drug solutions were added subsequently into
corresponding wells. The final concentrations of doxorubicin, vinblastine,
fluorouracil and mitoxantrone were 50 .mu.M, 100 .mu.M, 13.3 mM
and 5 .mu.M, respectively. After 2-hour incubation at 37.degree.
C., cell viability assays were performed as described in Example
7 and the buffer pH was measured.
Results
Krebs Ringer buffer at pH 6.8 was used to mimic the acidic extracellular
environment of solid tumor. At this incubation condition, the pH
raised by the urease activity was lower than what was observed in
FIG. 6D. However, the pH increase was sufficient to enhance the
antitumor efficacy of the two weak-base anticancer drugs tested.
At low urea level (2 mM), urease significantly enhanced the antitumor
activity of doxorubicin on A549 (FIG. 9A) and MDA-MB-231 cells (FIG.
9B), as well as that of vinblastine on MDA-MB-231 cells (FIG. 9B).
When urea level was increased to 8 mM, urease enhanced the activity
of both drugs on both cell lines (FIG. 9).
The antitumor efficacy of another weak-base anticancer drug, mitoxantrone,
was also significantly enhanced by urease when used to treat MDA-MB-231
cells (FIG. 10B) but not on A549 cells (FIG. 10A). Interestingly,
the activity of fluorouracil, which is not a weak-base drug, was
also enhanced by urease on A549 cells (FIG. 10A).
Although the invention has been described with respect to particular
embodiments, it will be apparent to those skilled in the art that
various changes.
Although the invention has been described with respect to particular
embodiments, it will be apparent to those skilled in the art that
various changes and modifications can be made without departing
from the invention.
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