U.S. patent application number 15/650317 was filed with the patent office on 2018-01-11 for cyclodextrin compositions encapsulating a selective atp inhibitor and uses thereof.
The applicant listed for this patent is The Johns Hopkins University. Invention is credited to Shanmugasundaram Ganapathy-Kanniappan, Jean-Francois Geschwind, Kenneth W. Kinzler, Surojit Sur, Bert Vogelstein.
Application Number | 20180008541 15/650317 |
Document ID | / |
Family ID | 53543385 |
Filed Date | 2018-01-11 |
United States Patent
Application |
20180008541 |
Kind Code |
A1 |
Geschwind; Jean-Francois ;
et al. |
January 11, 2018 |
Cyclodextrin Compositions Encapsulating a Selective ATP Inhibitor
and Uses Thereof
Abstract
The invention provides compositions comprising cyclodextrins
encapsulating a selective ATP inhibitor, as well as uses
thereof.
Inventors: |
Geschwind; Jean-Francois;
(Westport, CT) ; Ganapathy-Kanniappan;
Shanmugasundaram; (Baltimore, MD) ; Sur; Surojit;
(Gaithersburg, MD) ; Vogelstein; Bert; (Baltimore,
MD) ; Kinzler; Kenneth W.; (Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
Baltimore |
MD |
US |
|
|
Family ID: |
53543385 |
Appl. No.: |
15/650317 |
Filed: |
July 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14643603 |
Mar 10, 2015 |
9737487 |
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15650317 |
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PCT/US2015/011344 |
Jan 14, 2015 |
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14643603 |
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61992572 |
May 13, 2014 |
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61927259 |
Jan 14, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/02 20130101;
A61K 31/02 20130101; A61P 35/00 20180101; A61K 9/19 20130101; A61K
45/06 20130101; C08B 37/0012 20130101; A61K 47/6951 20170801; A61K
31/19 20130101; A61K 31/19 20130101; A61K 2300/00 20130101; C08B
37/0015 20130101; A61K 9/08 20130101; A61K 2300/00 20130101; A61K
9/146 20130101; C08L 5/16 20130101; A61K 47/40 20130101 |
International
Class: |
A61K 9/14 20060101
A61K009/14; C08B 37/16 20060101 C08B037/16; A61K 47/40 20060101
A61K047/40; A61K 31/02 20060101 A61K031/02; A61K 45/06 20060101
A61K045/06; A61K 9/08 20060101 A61K009/08; C08L 5/16 20060101
C08L005/16; A61K 31/19 20060101 A61K031/19 |
Goverment Interests
STATEMENT OF RIGHTS
[0002] This invention was made with government support under Grant
Numbers R01 CA160771, P30 CA006973, and NCRR UL1 RR 025005 awarded
by the National Institutes of Health. The U.S. government has
certain rights in the invention. This statement is included solely
to comply with 37 C.F.R. .sctn.401.14(a)(f)(4) and should not be
taken as an assertion or admission that the application discloses
and/or claims only one invention.
Claims
1. A composition comprising a cyclodextrin and a pharmaceutical
agent represented in the general formula: ##STR00005## wherein,
independently of each occurrence: X represents a halide, a
sulfonate, a carboxylate, an alkoxide, or an amine oxide; R.sub.1
represents OR, H, N(R'').sub.2, C1-C6 alkyl, C6-C12 aryl, C1-C6
heteroalkyl, or C6-C12 heteroaryl; R'' represents H, C1-C6 alkyl,
or C6-C12 aryl; R represents H, alkali metal, C1-C6 alkyl, C6-C12
aryl or C(O)R'; and R' represents H, C1-C20 alkyl or C6-C12 aryl,
wherein the cyclodextrin encapsulates the pharmaceutical agent.
2-30. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/927,259, filed on Jan. 14, 2014, and U.S.
Provisional Application No. 61/992,572, filed on May 13, 2014;
which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] The knowledge that cancer cells rely on increased glycolysis
rather than oxidative phosphorylation for survival is known as the
"Warburg hypothesis" (Warburg (1956) Science 123:309-314). This
concept constitutes the basis for using glycolysis and its
associated enzymes as unique targets for the development of new
anticancer therapeutic agents (Shaw (2006) Curr. Opin. Cell Biol.
18:598-608; Gatenby and Gillies (2007) Biochem. Cell Biol.
39:1358-1366). One such agent is 3-bromopyruvate (3-BrPA), a
synthetic brominated derivative of pyruvic acid that acts as an
irreversible glycolytic inhibitor (Ko et al. (2001) Cancer Lett.
173:83-91; Geschwind et al. (2002) Cancer Res. 62:3909-3913). It
disrupts energy metabolism by targeting the glycolytic enzyme,
glyceradehyde-3 phosphate dehydrogenase (GAPDH)
(Ganapathy-Kanniappan et al. (2009) Anticancer Res. 29:4909-4918).
Further, the anticancer effects of 3-BrPA have been consistent and
reproducible against multiple tumor models both in vitro and in
vivo. A wide variety of tumors have been demonstrated to be
sensitive to 3-BrPA treatment, including, for example, liver cancer
(Geschwind et al. (2002) Cancer Res. 62:3909-30913; Vali et al.
(2007) J. Vasc. Interv. Radiol. 18:95-101; and Ganapathy-Kanniappan
et al. (2012) Radiology 262:834-845), pancreatic cancer (Cao et al.
(2008) Clin. Cancer Res. 14:1831-1839; Bhardwaj et al. (2010)
Anticancer Res. 30:743-749; and Ota et al. (2013) Target Oncol.
8:145-151), brain tumor (El Sayed et al. (2012) J. Bioenerg.
Biomembr. 44:61-79; Davidescu et al. (2012) J. Bioenerg. Biomembr.
44:51-60) and breast cancer (Buijs et al. (2013) J. Vasc. Interv.
Radiol. 24:737-743). Together, the inhibition of GAPDH and the
molecular specificity of 3-BrPA have established that targeting
tumor glycolysis via 3-BrPA could be a viable strategy in treating
cancer, especially solid malignancies (Ganapathy-Kanniappan et al.
(2012) Oncotarget 3:940-95; Ganapathy-Kanniappan et al. (2013)
Anticancer Res. 33:13-20).
[0004] Despite the potential of selective ATP inhibitors, such as
3-halopyruvates like 3-BrPA, for therapeutic use, however, there
are several factors that have hampered development of systemic
administration formulations. For example, the alkylation (chemical)
properties of 3-halopyruvates and related compounds render them
very reactive with electrophilic molecules that has generally
required increases in dosing with the negative effect of increasing
toxicity, especially increased alkylation near the injection site.
In particular, the presence of water or any nucleophilic group,
such as amino or sulfhydryl groups commonly found in proteins,
chemically inactivates the compound. Also, the in vivo stability of
such compounds is influenced by multiple factors including
glutathione, NADH and other reducing molecules in the blood and
circulatory system. Hence, it is critical that the compounds remain
unaffected by such factors, at least until the first pass of
circulation.
[0005] While recognizing that protecting selective ATP inhibitors,
such as 3-halopyruvates like 3-BrPA, until they are delivered to
organs or tissues is critical for their antitumor efficacy under
systemic delivery, numerous approaches to achieve such protection,
such as encapsulating them in liposomes, microspheres, nanospheres,
nanoparticles, bubbles, and the like, have not been successful. For
example, it is known that molecules such as 3-BrPA undesirably
leach out rapidly from PEGylated liposomes or react with proteins
such as albumin in albumin-based nanoparticles. Although sporadic
reports have documented the intraperitoneal delivery of 3-BrPA in
preclinical models, the efficacy and dosage regimen were very
limited. Due to these failures, 3-BrPA therapies are currently
relegated to loco-regional delivery (e.g., percutaneous ablation,
intra-arterial delivery, and intra-tumoral injections) as opposed
to systemic delivery (Kunjithapatham et al. (2013) BMC Res. Notes
6:277).
[0006] Accordingly, there is a great need in the art to identify
compositions of selective ATP inhibitors, such as 3-halopyruvates
like 3-BrPA, suitable for systemic administration.
SUMMARY OF THE INVENTION
[0007] The present invention is based in part on the discovery that
encapsulating selective inhibitors of ATP production, such as
3-halopyruvates (e.g., 3-BrPA), within cyclodextrins both a)
stabilizes the alkylating agent in vivo by protecting the halogen
moiety away from aqueous and nucleophilic environments that would
deactivate the compound and b) provides a steady release of the
compound necessary to maintain a reasonable half-life of the
compound in vivo.
[0008] In one aspect, a composition comprising a cyclodextrin and a
pharmaceutical agent represented in the general formula:
##STR00001##
wherein, independently of each occurrence: X represents a halide, a
sulfonate, a carboxylate, an alkoxide, or an amine oxide; R.sub.1
represents OR, H, N(R'').sub.2, C1-C6 alkyl, C6-C12 aryl, C1-C6
heteroalkyl, or C6-C12 heteroaryl; R'' represents H, C1-C6 alkyl,
or C6-C12 aryl; R represents H, alkali metal, C1-C6 alkyl, C6-C12
aryl or C(O)R'; and R' represents H, C1-C20 alkyl or C6-C12 aryl,
wherein the cyclodextrin encapsulates the pharmaceutical agent, is
provided. In one embodiment, at least one .alpha.-D-glucopyranoside
unit of the cyclodextrin has at least one hydroxyl chemical group
replaced with an ionizable chemical group. In another embodiment,
the at least one hydroxyl chemical group of the at least one
.alpha.-D-glucopyranoside unit is selected from the group
consisting of C2, C3, and C6 hydroxyl chemical groups. In still
another embodiment, the C2, C3, and C6 hydroxyl chemical groups of
at least one .alpha.-D-glucopyranoside unit of the cyclodextrin
that are replaced with ionizable chemical groups. In yet another
embodiment, the at least one .alpha.-D-glucopyranoside unit of the
cyclodextrin is selected from the group consisting of two, three,
four, five, six, seven, eight, and all .alpha.-D-glucopyranoside
units of the cyclodextrin. In another embodiment, the ionizable
chemical group is the same at all replaced positions. In still
another embodiment, the ionizable chemical group is a weakly basic
functional group or a weakly acidic functional group. For example,
the weakly basic functional group (X) can have a pK.sub.a between
6.5 and 8.5 according to CH3-X.sup.- or the weakly acidic
functional group (Y) can have a pK.sub.a between 4.0 and 6.5
according to CH.sub.3-Y. In yet another embodiment, the weakly
basic or weakly acidic functional groups are selected from the
group consisting of amino, ethylene diamino, dimethyl ethylene
diamino, dimethyl anilino, dimethyl naphthylamino, succinyl,
carboxyl, sulfonyl, and sulphate functional groups. In another
embodiment, the cyclodextrin has a pK.sub.a1 of between 4.0 and
8.5. In still another embodiment, the composition is a liquid or
solid pharmaceutical formulation. In yet another embodiment, the
pharmaceutical agent is neutrally charged or hydrophobic. In
another embodiment, the cyclodextrin is selected from the group
consisting of .beta.-cyclodextrin, .alpha.-cyclodextrin, and
.gamma.-cyclodextrin. In still another embodiment, the cyclodextrin
is .beta.-cyclodextrin. In yet another embodiment, the
pharmaceutical agent is 3-halopyruvate. In another embodiment, the
pharmaceutical agent is 3-bromopyruvate. In still another
embodiment, the composition is formulated for systemic
administration. In yet another embodiment, the composition further
comprises an anti-cancer therapeutic agent.
[0009] In another aspect, a kit comprising a composition described
herein, and instructions for use, is provided.
[0010] In still another aspect, a method of treating a subject
having a cancer comprising administering to the subject a
therapeutically effective amount of a composition described herein,
is provided. In one embodiment, the composition is administered
systemically. In another embodiment, the systemic administration is
selected from the group consisting of oral, intravenous,
intraperitoneal, subcutaneous, and intramuscular administration. In
still another embodiment, the subject is treated with at least one
additional anti-cancer therapy. In yet another embodiment, the at
least one additional anti-cancer therapy is radiation therapy. In
another embodiment, the cancer is a solid tumor. In still another
embodiment, the cancer is selected from the group consisting of
liver cancer, pancreatic cancer, lung cancer and breast cancer. In
yet another embodiment, the cancer is liver cancer. In another
embodiment, the subject is a mammal. In still another embodiment,
the mammal is a human.
BRIEF DESCRIPTION OF FIGURES
[0011] FIG. 1 shows the chemical structure and toroidal topology of
beta-cyclodextrin molecule (see, for example Rasheed et al. (2008)
Sci. Pharm. 76: 567-598).
[0012] FIGS. 2A-2B show the effects of 3-BrPA or Beta-CD-3-BrPA on
MiaPaCa2 cells (FIG. 2A) and Suit-2 cells (FIG. 2B) after 24 hours
of treatment.
[0013] FIG. 3 shows the effects of 3-BrPA or Beta-CD-3-BrPA on
MiaPaCa2 cells after 72 hours of treatment.
[0014] FIG. 4 shows the effects of Beta-CD-3-BrPA on in vivo tumor
growth.
[0015] FIG. 5 shows complete tumor response in 3 mice treated with
Beta-CD-3-BrPA.
[0016] FIG. 6 shows the results of histopathological analysis of
the orthotopic MiaPaCa-2 tumors treated as in FIGS. 4 and 5.
[0017] FIG. 7 shows the results of NMR spectroscopy. The magnified
insert demonstrates the upfield shift of the methylene protons (0.1
ppm), which was observed upon complexation of .beta.-CD-3-BrPA.
[0018] FIG. 8 shows kill curves of 2D and 3D organotypic cell
cultures based on the luminescence-based cell viability in
MiaPaCa-2 (upper row) and Suit-2 (middle row) cells. Cells were
incubated under normoxic and hypoxic conditions for 72 hrs. prior
to exposure to 3-bromopyruvate (3-BrPA), 1:1-.beta.-Cyclodextrine
(CD)-3-BrPA, or .beta.-CD only as a control, for 24 hrs. Cells were
incubated for 24 hrs. before being treated with gemcitabine for 72
hrs. For the 3D organotypic cell cultures (lower row), lucMiaPaCa-2
cells were incubated under normoxic or hypoxic conditions for a
total of 6 days. Single-time treatments with 3-BrPA or
.beta.-CD-3-BrPA were performed on day 5 for 24 hrs. Exposure to
gemcitabine was initiated on day 3 for 72 hrs. Bioluminescence
imaging was performed on day 6 to evaluate drug penetration and
effects on cell viability. The lower-right box contains the
immune-blots for HIF-1alpha to confirm that hypoxia was
present.
[0019] FIG. 9 shows the effects of 3-bromopyruvate in 3D
organotypic cell culture. Homogeneous embedding lucMiaPaCa-2 cells
into the collagen I-matrix was confirmed by confocal light
microscopy (day 1). 3D organotypic cell cultures were incubated
under normoxic conditions and treated with 3-BrPA cumulatively
three times on alternate days. Phase-contrast microscopy and
bioluminescence imaging (the latter only for MiaPaCa-2 cells) were
performed on day 6 to evaluate effects on cell morphology and
viability. Immunofluorescence staining of F-actin and cleaved
caspase-3 were done in cryosections of the 3D organotypic cell
culture. DAPI was used as nucleic acid counterstain.
[0020] FIG. 10 shows further effects of 3-bromopyruvate in 3D
organotypic cell culture. Homogeneous embedding of Suit-2 cells
into the collagen I-matrix was confirmed by confocal light
microscopy (day 1). 3D organotypic cell cultures were incubated
under normoxic conditions and treated with 3-BrPA cumulatively
three times on alternate days. Phase-contrast microscopy and
bioluminescence imaging (the latter only for MiaPaCa-2 cells) were
performed on day 6 to evaluate effects on cell morphology and
viability. Immunofluorescence staining of F-actin and cleaved
caspase-3 were done in cryosections of the 3D organotypic cell
culture. DAPI was used as nucleic acid counterstain.
[0021] FIGS. 11A-11D show the effects of 3-bromopyruvate on cell
invasiveness. MiaPaCa-2 (FIG. 11A) and Suit-2 (FIG. 11B) cells were
plated into a Boyden invasion chamber. Incubation overnight was
followed by treatment with 3-bromopyruvate for 48 hrs. (MiaPaCa-2)
or 72 hrs. (Suit-2). Invaded cells on the bottom side of the
membrane of the invasion insert were stained using a Giemsa-like
staining. Images show invaded cells at 4.times., 10.times., and
20.times. magnification. Relative quantification of invasion was
calculated by measuring the area of stained cells in the entire
field of view at 10.times.. MMP-9 activity and secretion were
determined in the concentrated supernatant of MiaPaCa-2 and Suit-2
cells by zymography (FIG. 11C) and Western Blot (FIG. 11D). (*)
indicates statistical significant (p-value<0.05).
[0022] FIGS. 12A-12D show in vivo efficacy of
.beta.-cyclodextrin-3-bromopyruvate. A total of 42 male nude mice
were orthotopically implanted with a total of 1.5.times.10.sup.6
lucMiaPaCa-2 cells. After one week of xenograft growth, tumors were
confirmed using bioluminescence imaging (BLI). A representative
number of animals are shown in FIG. 12A. Animals were randomized to
receive .beta.-CD-3-BrPA (N=21), free 3-BrPA (N=7), gemcitabine
(N=7), and .beta.-CD (N=7). Animals were imaged once per week over
the course of 28 days. The overall progress of the signal is
demonstrated in FIG. 12B. According to Kaplan-Meier analysis,
animals treated with free 3-BrPA showed excessive treatment-related
toxicity leading to the loss of 5/7 animals at the end of the
experiment, such that a statistically relevant number did not
survive to be included in the final image analysis (FIG. 12C). The
vehicle-control (.beta.-CD) did not demonstrate any
treatment-related toxicity and was inert when given
intraperitoneally (FIG. 12C). Upon completion of the experiments,
all animals were sacrificed and exploratory necropsies were
performed in order to extract organs and to assess potential
damage. No organ toxicity (tissue effects) was observed for
.beta.-CD-3-BrPA, when compared with the inert vehicle (FIG.
12D).
[0023] FIG. 13 shows ex vivo pathological and immunohistochemical
tumor analysis. The H&E staining of tumors treated with
.beta.-CD, gemcitabine, or .beta.-CD-3-BrPA (3 representative
tumors are shown) demonstrated the treatment effects of
.beta.-CD-3-BrPA. The squares within the H&E-stained
whole-tumor overviews indicate the areas magnified for further
analysis of the anti-tumoral effects of the drugs, which was
confirmed by the staining for cleaved caspase-3 and Ki67. In
addition, the marked reduction of GAPDH as the primary target of
3-BrPA, as well as MCT-1 as the specific transporter, was
determined.
DETAILED DESCRIPTION OF THE INVENTION
[0024] It has been determined herein that cyclodextrins can
encapsulate selective inhibitors of ATP production such as
3-halopyruvates (e.g., 3-BrPA), in order to stabilize the
alkylating compound in an aqueous environment, as well as reduce
the ability of nucleophilic entities in proteins to access it,
thereby lowering its systemic toxicity and maintaining its
alkylating ability. Such compositions are demonstrated herein in
multiple in vitro cell lines, using different forms of
cyclodextrins (e.g., beta and alpha) and at different ratios of
active agent encapsulation relative to the cyclodextrin, and in in
vivo animal tumor models. For example, such compositions are
demonstrated herein to maintain the functional characteristics of
the selective inhibitors of ATP production to kill cancer cells
both in vitro and in vivo such that their activity can be preserved
and protected for systemic administration until it reaches the
target tissue, organ, and/or tumor while minimizing toxicity. This
determination was unexpected because cyclodextrins are known to
have a destabilizing effect on many compounds through direct
catalysis, particularly with increasing pH (Rasheed et al. (2008)
Sci. Pharm. 76: 567-598). Although this catalytic effect of
cyclodextrins would have been expected to be great for
3-halopyruvates since they are halogenated derivatives of pyruvic
acid, it was surprisingly determined that cyclodextrins actually
protected and stabilized 3-BrPA. It was further surprisingly
determined that cyclodextrins modified to replace one or more
hydroxyl groups on one or more of its .alpha.-D-glucopyranoside
units with ionizable groups resulting in negative charges (anions)
stabilizes the 3-halopyruvates better than those having ionizable
groups resulting in positive charges (cations) or unmodified
cyclodextrins, such as unmodified alpha- or beta-cyclodextrin.
Without being bound by theory, it is believed that anionic moieties
on cyclodextrins force the halogen atom (e.g., bromine) of a
halopyruvate (e.g., 3-BrPA) to sit in the cavity. It was also
surprisingly determined that .beta.-cyclodextrins encapsulate
3-BrPA in a form that protects and stabilizes 3-BrPA for in vivo
efficacy especially and also in vitro efficacy significantly better
than .alpha.-cyclodextrins.
[0025] Thus, the present invention provides compositions and kits
comprising such 3-halopyruvate compounds encapsulated within
cyclodextrins, as well as methods of making and using such
compositions and kits.
A. Definitions
[0026] In order for the present invention to be more readily
understood, certain terms and phrases are defined below and
throughout the specification.
[0027] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0028] The term "3-bromopyruvate" or "3-BrPA" refers to
3-bromopyruvate, analogs and derivatives of 3-brompyruvate,
prodrugs of 3-bromopyruvate, metabolites of 3-bromopyruvate and
salts thereof.
[0029] The term "administering" means providing a pharmaceutical
agent or composition to a subject, and includes, but is not limited
to, administering by a medical professional and
self-administering.
[0030] The term "cancer" includes, but is not limited to, solid
tumors and blood borne tumors. The term cancer includes diseases of
the skin, tissues, organs, bone, cartilage, blood and vessels. The
term "cancer" further encompasses primary and metastatic
cancers.
[0031] The term "inhibit" or "inhibits" means to decrease,
suppress, attenuate, diminish, arrest, or stabilize the development
or progression of a disease, disorder, or condition, the activity
of a biological pathway, or a biological activity, such as the
growth of a solid malignancy, e.g., by at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% compared to an
untreated control subject, cell, biological pathway, or biological
activity or compared to the target, such as a growth of a solid
malignancy, in a subject before the subject is treated. By the term
"decrease" is meant to inhibit, suppress, attenuate, diminish,
arrest, or stabilize a symptom of a cancer disease, disorder, or
condition. It will be appreciated that, although not precluded,
treating a disease, disorder or condition does not require that the
disease, disorder, condition or symptoms associated therewith be
completely eliminated.
[0032] The term "modulation" refers to upregulation (i.e.,
activation or stimulation), downregulation (i.e., inhibition or
suppression) of a response, or the two in combination or apart.
[0033] The term "pharmaceutically acceptable" is employed herein to
refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0034] The term "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient, or
solvent encapsulating material, involved in carrying or
transporting the subject compound from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters,
polycarbonates and/or polyanhydrides; and (22) other non-toxic
compatible substances employed in pharmaceutical formulations.
[0035] The term "pharmaceutically-acceptable salts" refers to the
relatively non-toxic, inorganic and organic salts of compounds.
[0036] A "subject" can include a human subject for medical
purposes, such as for the treatment of an existing disease,
disorder, condition or the prophylactic treatment for preventing
the onset of a disease, disorder, or condition or an animal subject
for medical, veterinary purposes, or developmental purposes.
Suitable animal subjects include mammals including, but not limited
to, primates, e.g., humans, monkeys, apes, gibbons, chimpanzees,
orangutans, macaques and the like; bovines, e.g., cattle, oxen, and
the like; ovines, e.g., sheep and the like; caprines, e.g., goats
and the like; porcines, e.g., pigs, hogs, and the like; equines,
e.g., horses, donkeys, zebras, and the like; felines, including
wild and domestic cats; canines, including dogs; lagomorphs,
including rabbits, hares, and the like; and rodents, including
mice, rats, guinea pigs, and the like. An animal may be a
transgenic animal. In some embodiments, the subject is a human
including, but not limited to, fetal, neonatal, infant, juvenile,
and adult subjects. Further, a "subject" can include a patient
afflicted with or suspected of being afflicted with a disease,
disorder, or condition. Thus, the terms "subject" and "patient" are
used interchangeably herein. Subjects also include animal disease
models (e.g., rats or mice used in experiments, and the like).
[0037] The terms "prevent," "preventing," "prevention,"
"prophylactic treatment," and the like refer to reducing the
probability of developing a disease, disorder, or condition in a
subject, who does not have, but is at risk of or susceptible to
developing a disease, disorder, or condition.
[0038] The term "subject suspected of having" means a subject
exhibiting one or more clinical indicators of a disease or
condition. In certain embodiments, the disease or condition is
cancer. In certain embodiments, the cancer is leukemia or
lymphoma.
[0039] The term "subject in need thereof" means a subject
identified as in need of a therapy or treatment.
[0040] The terms "systemic administration," "administered
systemically," "peripheral administration," and "administered
peripherally" mean the administration of a compound, drug or other
material other than directly into the central nervous system, such
that it enters the patient's system and, thus, is subject to
metabolism and other like processes, for example, subcutaneous
administration.
[0041] The term "therapeutic agent" or "pharmaceutical agent"
refers to an agent capable of having a desired biological effect on
a host. Chemotherapeutic and genotoxic agents are examples of
therapeutic agents that are generally known to be chemical in
origin, as opposed to biological, or cause a therapeutic effect by
a particular mechanism of action, respectively. Examples of
therapeutic agents of biological origin include growth factors,
hormones, and cytokines. A variety of therapeutic agents is known
in the art and may be identified by their effects. Certain
therapeutic agents are capable of regulating red cell proliferation
and differentiation. Examples include chemotherapeutic nucleotides,
drugs, hormones, non-specific (e.g. non-antibody) proteins,
oligonucleotides (e.g., antisense oligonucleotides that bind to a
target nucleic acid sequence (e.g., mRNA sequence)), peptides, and
peptidomimetics.
[0042] The term "therapeutic effect" refers to a local or systemic
effect in animals, particularly mammals, and more particularly
humans, caused by a pharmacologically active substance. The term
thus means any substance intended for use in the diagnosis, cure,
mitigation, treatment or prevention of disease or in the
enhancement of desirable physical or mental development and
conditions in an animal or human. The phrase
"therapeutically-effective amount" means that amount of such a
substance that produces some desired local or systemic effect at a
reasonable benefit/risk ratio applicable to any treatment. In
certain embodiments, a therapeutically effective amount of a
compound will depend on its therapeutic index, solubility, and the
like. For example, certain compounds discovered by the methods of
the present invention may be administered in a sufficient amount to
produce a reasonable benefit/risk ratio applicable to such
treatment.
[0043] The terms "therapeutically-effective amount" and "effective
amount" as used herein means that amount of a compound, material,
or composition comprising a compound of the present invention which
is effective for producing some desired therapeutic effect in at
least a sub-population of cells in an animal at a reasonable
benefit/risk ratio applicable to any medical treatment.
[0044] The term "treating" a disease in a subject or "treating" a
subject having a disease refers to subjecting the subject to a
pharmaceutical treatment, e.g., the administration of a drug, such
that at least one symptom of the disease is decreased or prevented
from worsening.
[0045] The terms "tumor," "solid malignancy," or "neoplasm" refer
to a lesion that is formed by an abnormal or unregulated growth of
cells. Preferably, the tumor is malignant, such as that formed by a
cancer.
B. Cyclodextrins
[0046] The term "cyclodextrin" refers to a family of cyclic
oligosaccharides composed of 5 or more .alpha.-D-glucopyranoside
units linked together by C1-C4 bonds having a toroidal topological
structure, wherein the larger and the smaller openings of the
toroid expose certain hydroxyl groups of the
.alpha.-D-glucopyranoside units to the surrounding environment
(e.g., solvent) (see, for examples, FIG. 1). The term "inert
cyclodextrin" refers to a cyclodextrin containing
.alpha.-D-glucopyranoside units having the basic formula
C.sub.6H.sub.12O.sub.6 and glucose structure without any additional
chemical substitutions (e.g., .alpha.-cyclodextrin having 6 glucose
monomers, .beta.-cyclodextrin having 7 glucose monomers, and
.gamma.-cyclodextrin having 8 glucose monomers). The term
"cyclodextrin internal phase" refers to the relatively less
hydrophilic region enclosed within (i.e., encapsulated by) the
toroid topology of the cyclodextrin structure. The term
"cyclodextrin external phase" refers to the region not enclosed by
the toroid topology of the cyclodextrin structure and can include,
for example, the aqueous environment present during systemic
administration in vivo or to the internal phase of a structure that
itself encapsulates the selective ATP production
inhibitor/cyclodextrin complex. Cyclodextrins are useful for
solubilizing hydrophobic compositions (see, for example, Albers and
Muller (1995) Crit. Rev. Therap. Drug Carrier Syst. 12:311-337;
Zhang and Ma (2013) Adv. Drug Delivery Rev. 65:1215-1233;
Laza-Knoerr et al. (2010) J. Drug Targ. 18:645-656; Challa et al.
(2005) AAPS PharmSci. Tech. 6:E329-357; Uekama et al. (1998) Chem.
Rev. 98:2045-2076; Szejtli (1998) Chem. Rev. 98:1743-1754; Stella
and He (2008) Toxicol. Pathol. 36:30-42; Rajewski and Stella (1996)
J. Pharm. Sci. 85:1142-1169; Thompson (1997) Crit. Rev. Therap.
Drug Carrier Sys. 14:1-104; and Irie and Uekama (1997) J. Pharm.
Sci. 86:147-162). Any substance located within the cyclodextrin
internal phase is said to be "encapsulated."
[0047] As used herein, a cyclodextrin is useful according to the
present invention so long as the cyclodextrins can encapsulate a
selective ATP production inhibitor. In some embodiments, the
cyclodextrin further bears ionizable (e.g., weakly basic and/or
weakly acidic) functional groups to enhance the stabilization of
the selective ATP production inhibitor. By protecting the stability
of the selective ATP production inhibitor, it is meant that the
selective ATP production inhibitor/cyclodextrin complex makes the
selective ATP production inhibitor molecule more stable as seen by
photo stability, shelf life stability, thermal stability, stability
against intramolecular cyclization, stability to acid hydrolysis,
stability against general degradation, and the like, as compared to
the stability of a selective ATP production inhibitor molecule that
is not in a complex with cyclodextrin.
[0048] For encapsulating a desired therapeutic agent, cyclodextrins
can be selected and/or chemically modified according to the
characteristics of the desired therapeutic agent and parameters for
efficient, high-concentration loading therein. For example, it is
preferable that the cyclodextrin itself have high solubility in
water in order to facilitate loading of a therapeutic agent, such
as a 3-halopyruvate. In some embodiments, the water solubility of
the cyclodextrin is at least 10 mg/mL, 20 mg/mL, 30 mg/mL, 40
mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL
or higher. Methods for achieving such enhanced water solubility are
well known in the art.
[0049] In some embodiments, a large association constant with the
therapeutic agent is preferable and can be obtained by selecting
the number of glucose units in the cyclodextrin based on the size
of the therapeutic agent (see, for example, Albers and Muller
(1995) Crit. Rev. Therap. Drug Carrier Syst. 12:311-337; Stella and
He (2008) Toxicol. Pathol. 36:30-42; and Rajewski and Stella (1996)
J. Pharm. Sci. 85:1142-1169). As a result, the solubility (nominal
solubility) of the therapeutic agent in the presence of
cyclodextrin can be further improved. For example, the association
constant of the cyclodextrin with the therapeutic agent can be 100,
200, 300, 400, 500, 600, 700, 800, 900, 1,000, or higher.
[0050] Derivatives formed by reaction with cyclodextrin hydroxyl
groups (e.g., those lining the upper and lower ridges of the toroid
of an inert cyclodextrin) are readily prepared and offer a means of
modifying the physicochemical properties of the parent (inert)
cyclodextrin. In some embodiments, the physicochemical properties
of the inert cyclodextrin molecule or cyclodextrin molecule that is
not complexed with a selective ATP production inhibitor differ from
the properties of a cyclodextrin molecule complexed with the
selective ATP production inhibitor. Accordingly, the selective ATP
production inhibitor molecules complexed with cyclodextrin can be
characterized by observing changes in solubility, chemical
reactivity, UV/VIS absorbance, drug retention, chemical stability,
and the like. For example, it has been determined herein that
modifying hydroxyl groups, such as those facing away from the
cyclodextrin interior phase, can be replaced with ionizable
chemical groups to facilitate loading of therapeutic agents, such
as poorly soluble or hydrophobic agents, within the modified
cyclodextrins and stabilization thereof. In one embodiment, a
modified cyclodextrin having at least one hydroxyl group
substituted with an ionizable chemical group will result in a
charged moiety under certain solvent (e.g., pH) conditions. The
term "charged cyclodextrin" refers to a cyclodextrin having one or
more of its hydroxyl groups substituted with a charged moiety and
the moiety bearing a charge. Such a moiety can itself be a charged
group or it can comprise an organic moiety (e.g., a Ci-C.sub.6
alkyl or Ci-C.sub.6 alkyl ether moiety) substituted with one or
more charged moieties.
[0051] In one embodiment, the "ionizable" or "charged" moieties are
weakly ionizable. Weakly ionizable moieties are those that are
either weakly basic or weakly acidic. Weakly basic functional
groups (X) have a pK.sub.a of between about 6.0-9.0, 6.5-8.5,
7.0-8.0, 7.5-8.0, and any range in between inclusive according to
CH.sub.3-X. Similarly, weakly acidic functional groups (Y) have a
log dissociation constant (pK.sub.a) of between about 3.0-7.0,
4.0-6.5, 4.5-6.5, 5.0-6.0, 5.0-5.5, and any range in between
inclusive according to CH.sub.3-Y. The pKa parameter is a
well-known measurement of acid/base properties of a substance and
methods for pKa determination are conventional and routine in the
art. For example, the pKa values for many weak acids are tabulated
in reference books of chemistry and pharmacology. See, for example,
IUPAC Handbook of Pharmaceutical Salts, ed. by P. H. Stahl and C. G
Wermuth, Wiley-VCH, 2002; CRC Handbook of Chemistry and Physics,
82nd Edition, ed. by D. R. Lide, CRC Press, Florida, 2001, p. 8-44
to 8-56. Since cyclodextrins with more than one ionizable group
have pKa of the second and subsequent groups each denoted with a
subscript.
[0052] Representative anionic moieties include, without any
limitation, succinyl, carboxylate, carboxymethyl, sulfonyl,
phosphate, sulfoalkyl ether, sulphate carbonate, thiocarbonate,
thiocarbonate, phosphate, phosphonate, sulfonate, nitrate, and
borate groups.
[0053] Representative cationic moieties include, without
limitation, amino, guanidine, and quaternary ammonium groups.
[0054] In another embodiment, the modified cyclodextrin is a
"polyanion" or "polycation." A polyanion is a modified cyclodextrin
having more than one negatively charged group resulting in net
negative ionic charger of more than two units. A polycation is a
modified cyclodextrin having more than one positively charged group
resulting in net positive ionic charger of more than two units.
[0055] In another embodiment, the modified cyclodextrin is a
"chargeable amphiphile." By "chargeable" is meant that the
amphiphile has a pK in the range pH 4 to pH 8 or 8.5. A chargeable
amphiphile may therefore be a weak acid or base. By "amphoteric"
herein is meant a modified cyclodextrin having a ionizable groups
of both anionic and cationic character wherein: 1) at least one,
and optionally both, of the cation and anionic amphiphiles is
chargeable, having at least one charged group with a pK between 4
and 8 to 8.5, 2) the cationic charge prevails at pH 4, and 3) the
anionic charge prevails at pH 8 to 8.5.
[0056] In some embodiments, the "ionizable" or "charged"
cyclodextrins as a whole, whether polyionic, amphiphilic, or
otherwise, are weakly ionizable (i.e., have a pKa.sub.1 of between
about 4.0-8.5, 4.5-8.0, 5.0-7.5, 5.5-7.0, 6.0-6.5, and any range in
between inclusive).
[0057] Any one, some, or all hydroxyl groups of any one, some or
all .alpha.-D-glucopyranoside units of a cyclodextrin can be
modified to an ionizable chemical group as described herein. Since
each cyclodextrin hydroxyl group differs in chemical reactivity,
reaction with a modifying moiety can produce an amorphous mixture
of positional and optical isomers. Alternatively, certain chemistry
can allow for pre-modified .alpha.-D-glucopyranoside units to be
reacted to form uniform products.
[0058] The aggregate substitution that occurs is described by a
term called the degree of substitution. For example, a
6-ethylenediamino-.beta.-cyclodextrin with a degree of substitution
of seven would be composed of a distribution of isomers of
6-ethylenediamino-.beta.-cyclodextrin in which the average number
of ethylenediamino groups per 6-ethylenediamino-.beta.-cyclodextrin
molecule is seven. Degree of substitution can be determined by mass
spectrometry or nuclear magnetic resonance spectroscopy.
Theoretically, the maximum degree of substitution is 18 for
.alpha.-cyclodextrin, 21 for .beta., and 24 for
.gamma.-cyclodextrin, however, substituents themselves having
hydroxyl groups present the possibility for additional
hydroxylalkylations. The degree of substitution can be 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or more and can encompass complete substitution.
[0059] Another parameter is the stereochemical location of a given
hydroxyl substitution. In one embodiment, at least one hydroxyl
facing away from the cyclodextrin interior is substituted with an
ionizable chemical group. For example, the C2, C3, C6, C2 and C3,
C2 and C6, C3 and C6, and all three of C2-C3-C6 hydroxyls of at
least one .alpha.-D-glucopyranoside unit are substituted with an
ionizable chemical group. Such carbon positions are well known in
the art. For example, the CH2OH moiety shown in FIG. 1 of each
.alpha.-D-glucopyranoside unit represents the C6 carbon. Any such
combination of hydroxyls can similarly be combined with at least
two, three, four, five, six, seven, eight, nine, ten, eleven, up to
all of the .alpha.-D-glucopyranoside units in the modified
cyclodextrin as well as in combination with any degree of
substitution described herein.
[0060] It is also acceptable to combine one or more of the
cyclodextrins described herein.
C. Selective Inhibitors of ATP Production and Related Compounds
[0061] Some embodiments of the present invention relate to the
encapsulation of selective inhibitors of ATP production within
cyclodextrins. The term "selective inhibitors of ATP production"
refers to anti-metabolite agents that inhibit ATP production by
interfering with the enzymatic process of generating ATP (e.g.,
GAPDH inhibitors such as 3-halopyruvates like 3-bromopyruvate). In
some embodiments, the selective inhibitor of ATP production is an
"antineoplastic alkylating agent," which refers to an agent used in
cancer treatment that causes replacement of hydrogen by an alkyl
group. As used herein the term "alkyl" refers to C.sub.1-20
inclusive, linear (i.e., "straight-chain"), branched, or cyclic,
saturated or at least partially and in some cases fully unsaturated
(i.e., alkenyl and alkynyl) hydrocarbon radicals derived from a
hydrocarbon moiety containing between one and twenty carbon atoms
by removal of a single hydrogen atom. Representative alkyl groups
include, but are not limited to, methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,
sec-pentyl, iso-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl,
n-octyl, n-decyl, n-undecyl, dodecyl, and the like, ethenyl,
propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl,
propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups.
"Branched" refers to an alkyl group in which a lower alkyl group,
such as methyl, ethyl or propyl, is attached to a linear alkyl
chain. "Lower alkyl" refers to an alkyl group having 1 to about 8
carbon atoms (i.e., a C.sub.1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7,
or 8 carbon atoms. "Higher alkyl" refers to an alkyl group having
about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl"
refers, in particular, to C.sub.1-8 straight-chain alkyls. In other
embodiments, "alkyl" refers, in particular, to C.sub.1-8
branched-chain alkyls.
[0062] Alkyl groups can optionally be substituted (a "substituted
alkyl") with one or more alkyl group substituents, which can be the
same or different. The term "alkyl group substituent" includes but
is not limited to alkyl, substituted alkyl, halo, arylamino, acyl,
hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl,
aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There
can be optionally inserted along the alkyl chain one or more
oxygen, sulfur or substituted or unsubstituted nitrogen atoms,
wherein the nitrogen substituent is hydrogen, lower alkyl (also
referred to herein as "alkylaminoalkyl"), or aryl. Thus, as used
herein, the term "substituted alkyl" includes alkyl groups, as
defined herein, in which one or more atoms or functional groups of
the alkyl group are replaced with another atom or functional group,
including for example, alkyl, substituted alkyl, halogen, aryl,
substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,
dialkylamino, sulfate, and mercapto.
[0063] In one embodiment, selective inhibitors of ATP production
are generally represented by the formula:
##STR00002##
[0064] wherein X represents a halide, a sulfonate, a carboxylate,
an alkoxide, or an amine oxide. In certain embodiments, X is a
halide selected from the group consisting of: fluoride, bromide,
chloride, and iodide. In one embodiment, the inhibitor is a
3-halopyruvate. In certain other embodiments, the 3-halopyruvate is
selected from the group consisting of: 3-fluoropyruvate,
3-chloropyruvate, 3-bromopyruvate and 3-iodopyruvate. In one
embodiment, the 3-halopyruvate is 3-bromopyruvate. In other
embodiments, X is a sulfonate and may be selected from the group
consisting of: triflate, mesylate and tosylate. In yet another
embodiment, X is an amine oxide is dimethylamine oxide. In certain
embodiments R.sub.1 represents OR, H, N(R'').sub.2, C1-C6 alkyl,
C6-C12 aryl, C1-C6 heteroalkyl, or a C6-C12 heteroaryl.
Independently, in other embodiments, R'' represents H, C1-C6 alkyl,
or C6-C12 aryl. Independently, in still other embodiments, R
represents H, alkali metal, C1-C6 alkyl, C6-C12 aryl or C(O)R'; and
R' represents H, C1-C20 alkyl or C6-C12 aryl.
[0065] In a preferred embodiment, the invention further provides
inhibitors of ATP production represented by general formula:
X--CH2-CO--COOH,
[0066] wherein X represents a halide, a sulfonate, a carboxylate,
an alkoxide, or an amine oxide. In certain embodiments, X is a
halide and may be selected from the group consisting of: fluoride,
bromide, chloride, and iodide. In one embodiment, the inhibitor is
3-halopyruvate. In certain embodiments, the 3-halopyruvate is
selected from the group consisting of: 3-fluoropyruvate,
3-chloropyruvate, 3-bromopyruvate and 3-iodopyruvate. In one
embodiment, the 3-halopyruvate is 3-bromopyruvate. In other
embodiments, X is a sulfonate selected from the group consisting
of: triflate, mesylate and tosylate. In yet another embodiment, X
is an amine oxide is dimethylamine oxide.
[0067] Other analogs, derivatives, prodrugs, metabolites and salts
thereof of 3-bromopyruvate can also be used, provided that these
compounds or compositions have an anticancer effect that is
statistically similar to that of 3-bromopyruvate. When referring
herein to a treatment using 3-bromopyruvate, it should be
understood that the treatment may also be conducted with analogs,
derivatives, prodrugs, metabolites and salts of 3-bromopyruvate,
where applicable.
D. Cyclodextrin/ATP Inhibitor Compositions
[0068] The present invention provides pharmaceutical compositions
comprising selective inhibitors of ATP production described above
encapsulated within inert and/or modified cyclodextrins. Such
complexes are referred to herein as cyclodextrin/ATP inhibitor
compositions. The ratio of selective inhibitor of ATP production to
cyclodextrin may be 1:1 such that one inhibitor molecule forms a
complex with one cyclodextrin molecule. Alternatively, the ratio
can be 2:1, 3:1, 4:1, 5:1, or more.
[0069] In one aspect, the present invention provides
pharmaceutically acceptable compositions which comprise a
therapeutically-effective amount of one or more such
cyclodextrin/ATP inhibitors described above, formulated together
with one or more pharmaceutically acceptable carriers (additives)
and/or diluents. In another aspect the compositions can be
administered as such or in admixtures with pharmaceutically
acceptable carriers and can also be administered in conjunction
with other anti-cancer therapies, such as chemotherapeutic agents,
scavenger compounds, radiation therapy, biologic therapy, and the
like. Conjunctive therapy thus includes sequential, simultaneous
and separate, or co-administration of the composition, wherein the
therapeutic effects of the first administered has not entirely
disappeared when the subsequent compound is administered.
[0070] As described in detail below, the pharmaceutical
compositions of the present invention may be specially formulated
for administration in solid or liquid form, including those adapted
for the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets, e.g.,
those targeted for buccal, sublingual, and systemic absorption,
boluses, powders, granules, pastes for application to the tongue;
(2) parenteral administration, for example, by subcutaneous,
intramuscular, intravenous or epidural injection as, for example, a
sterile solution or suspension, or sustained-release formulation;
(3) topical application, for example, as a cream, ointment, or a
controlled-release patch or spray applied to the skin; (4)
intravaginally or intrarectally, for example, as a pessary, cream
or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)
nasally.
[0071] As set out above, certain embodiments of the selective ATP
inhibitors or cyclodextrin/ATP inhibitor compositions may contain a
basic functional group, such as amino or alkylamino, and are, thus,
capable of forming pharmaceutically-acceptable salts with
pharmaceutically-acceptable acids. These salts can be prepared in
situ in the administration vehicle or the dosage form manufacturing
process, or by separately reacting a purified compound of the
invention in its free base form with a suitable organic or
inorganic acid, and isolating the salt thus formed during
subsequent purification. Representative salts include the
hydrobromide, hydrochloride, sulfate, bisulfate, phosphate,
nitrate, acetate, valerate, oleate, palmitate, stearate, laurate,
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, napthylate, mesylate, glucoheptonate,
lactobionate, and laurylsulphonate salts and the like (see, for
example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.
66:1-19).
[0072] The pharmaceutically acceptable salts of the subject
compounds include the conventional nontoxic salts or quaternary
ammonium salts of the compounds, e.g., from non-toxic organic or
inorganic acids. For example, such conventional nontoxic salts
include those derived from inorganic acids such as hydrochloride,
hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like;
and the salts prepared from organic acids such as acetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic,
oxalic, isothionic, and the like.
[0073] In other cases, the selective ATP inhibitors or
cyclodextrin/ATP inhibitor compositions of the present invention
may contain one or more acidic functional groups and, thus, are
capable of forming pharmaceutically-acceptable salts with
pharmaceutically-acceptable bases. These salts can likewise be
prepared in situ in the administration vehicle or the dosage form
manufacturing process, or by separately reacting the purified
compound in its free acid form with a suitable base, such as the
hydroxide, carbonate or bicarbonate of a
pharmaceutically-acceptable metal cation, with ammonia, or with a
pharmaceutically-acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like (see, for example, Berge et al., supra).
[0074] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0075] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0076] Cyclodextrin/ATP inhibitor composition formulations include
those suitable for oral, nasal, topical (including buccal and
sublingual), rectal, vaginal and/or parenteral administration. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any methods well known in the art of pharmacy.
The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the host being treated and the particular mode of
administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the compound which produces a
therapeutic effect.
[0077] In certain embodiments, a formulation of cyclodextrin/ATP
inhibitor compositions can comprise other carriers to allow more
stability, to allow more stability, different releasing properties
in vivo, targeting to a specific site, or any other desired
characteristic that will allow more effective delivery of the
complex to a subject or a target in a subject, such as, without
limitation, liposomes, microspheres, nanospheres, nanoparticles,
bubbles, micelle forming agents, e.g., bile acids, and polymeric
carriers, e.g., polyesters and polyanhydrides. In certain
embodiments, an aforementioned formulation renders orally
bioavailable a compound of the present invention.
[0078] Liquid dosage formulations of cyclodextrin/ATP inhibitor
compositions include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0079] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0080] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0081] Formulations suitable for oral administration may be in the
form of capsules, cachets, pills, tablets, lozenges (using a
flavored basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or
non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia) and/or
as mouth washes and the like, each containing a predetermined
amount of an active ingredient. A cyclodextrin/ATP inhibitor
composition of the present invention may also be administered as a
bolus, electuary or paste.
[0082] In solid dosage forms (e.g., capsules, tablets, pills,
dragees, powders, granules and the like), the active ingredient is
mixed with one or more pharmaceutically-acceptable carriers, such
as sodium citrate or dicalcium phosphate, and/or any of the
following: (1) fillers or extenders, such as starches, lactose,
sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such
as, for example, carboxymethylcellulose, alginates, gelatin,
polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such
as glycerol; (4) disintegrating agents, such as agar-agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate; (5) solution retarding agents,
such as paraffin; (6) absorption accelerators, such as quaternary
ammonium compounds; (7) wetting agents, such as, for example, cetyl
alcohol, glycerol monostearate, and non-ionic surfactants; (8)
absorbents, such as kaolin and bentonite clay; (9) lubricants, such
a talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate, and mixtures thereof; and (10)
coloring agents. In the case of capsules, tablets and pills, the
compositions may also comprise buffering agents. Solid compositions
of a similar type may also be employed as fillers in soft and
hard-shelled gelatin capsules using such excipients as lactose or
milk sugars, as well as high molecular weight polyethylene glycols
and the like.
[0083] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0084] The tablets, and other solid dosage forms, such as dragees,
capsules, pills and granules, may optionally be scored or prepared
with coatings and shells, such as enteric coatings and other
coatings well known in the pharmaceutical-formulating art. They may
also be formulated so as to provide slow or controlled release of
the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. Compositions may also be formulated for rapid
release, e.g., freeze-dried. They may be sterilized by, for
example, filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved in sterile water, or some other
sterile injectable medium immediately before use. These
compositions may also optionally contain opacifying agents and may
be of a composition that they release the active ingredient(s)
only, or preferentially, in a certain portion of the
gastrointestinal tract, optionally, in a delayed manner. Examples
of embedding compositions which can be used include polymeric
substances and waxes. The active ingredient can also be in
micro-encapsulated form, if appropriate, with one or more of the
above-described excipients.
[0085] Formulations for rectal or vaginal administration may be
presented as a suppository, which may be prepared by mixing one or
more compounds of the invention with one or more suitable
nonirritating excipients or carriers comprising, for example, cocoa
butter, polyethylene glycol, a suppository wax or a salicylate, and
which is solid at room temperature, but liquid at body temperature
and, therefore, will melt in the rectum or vaginal cavity and
release the active compound.
[0086] Formulations which are suitable for vaginal administration
also include pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing such carriers as are known in the art
to be appropriate.
[0087] Dosage forms for the topical or transdermal administration
of a cyclodextrin/ATP inhibitor composition of the present
invention include powders, sprays, ointments, pastes, creams,
lotions, gels, solutions, patches and inhalants. The active
compound may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0088] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0089] Powders and sprays can contain excipients such as lactose,
talc, silicic acid, aluminum hydroxide, calcium silicates and
polyamide powder, or mixtures of these substances. Sprays can
additionally contain customary propellants, such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,
such as butane and propane.
[0090] Transdermal patches have the added advantage of providing
controlled delivery to the body. Such dosage forms can be made by
dissolving or dispersing the compound in the proper medium.
Absorption enhancers can also be used to increase the flux of the
compound across the skin. The rate of such flux can be controlled
by either providing a rate controlling membrane or dispersing the
compound in a polymer matrix or gel.
[0091] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0092] Pharmaceutical compositions suitable for parenteral
administration can comprise sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
sugars, alcohols, antioxidants, buffers, bacteriostats, solutes
which render the formulation isotonic with the blood of the
intended recipient or suspending or thickening agents.
[0093] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0094] In certain embodiments, the above-described pharmaceutical
compositions can be combined with other pharmacologically active
compounds ("second active agents") known in the art according to
the methods and compositions provided herein. Second active agents
can be large molecules (e.g., proteins) or small molecules (e.g.,
synthetic inorganic, organometallic, or organic molecules). In one
embodiment, second active agents independently or synergistically
help to treat cancer.
[0095] For example, chemotherapeutic agents are anti-cancer agents.
The term chemotherapeutic agent includes, without limitation,
platinum-based agents, such as carboplatin and cisplatin; nitrogen
mustard alkylating agents; nitrosourea alkylating agents, such as
carmustine (BCNU) and other alkylating agents; antimetabolites,
such as methotrexate; purine analog antimetabolites; pyrimidine
analog antimetabolites, such as fluorouracil (5-FU) and
gemcitabine; hormonal antineoplastics, such as goserelin,
leuprolide, and tamoxifen; natural antineoplastics, such as taxanes
(e.g., docetaxel and paclitaxel), aldesleukin, interleukin-2,
etoposide (VP-16), interferon alfa, and tretinoin (ATRA);
antibiotic natural antineoplastics, such as bleomycin,
dactinomycin, daunorubicin, doxorubicin, and mitomycin; and vinca
alkaloid natural antineoplastics, such as vinblastine and
vincristine.
[0096] Further, the following drugs may also be used in combination
with an antineoplastic agent, even if not considered antineoplastic
agents themselves: dactinomycin; daunorubicin HCl; docetaxel;
doxorubicin HCl; epoetin alfa; etoposide (VP-16); ganciclovir
sodium; gentamicin sulfate; interferon alfa; leuprolide acetate;
meperidine HCl; methadone HCl; ranitidine HCl; vinblastin sulfate;
and zidovudine (AZT). For example, fluorouracil has recently been
formulated in conjunction with epinephrine and bovine collagen to
form a particularly effective combination.
[0097] Still further, the following listing of amino acids,
peptides, polypeptides, proteins, polysaccharides, and other large
molecules may also be used: interleukins 1 through 18, including
mutants and analogues; interferons or cytokines, such as
interferons .alpha., .beta., and .gamma.; hormones, such as
luteinizing hormone releasing hormone (LHRH) and analogues and,
gonadotropin releasing hormone (GnRH); growth factors, such as
transforming growth factor-.beta. (TGF-.beta.), fibroblast growth
factor (FGF), nerve growth factor (NGF), growth hormone releasing
factor (GHRF), epidermal growth factor (EGF), fibroblast growth
factor homologous factor (FGFHF), hepatocyte growth factor (HGF),
and insulin growth factor (IGF); tumor necrosis factor-.alpha.
& .beta. (TNF-.alpha. & .beta.); invasion inhibiting
factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7);
somatostatin; thymosin-.alpha.-1; .gamma.-globulin; superoxide
dismutase (SOD); complement factors; anti-angiogenesis factors;
antigenic materials; and pro-drugs.
[0098] Chemotherapeutic agents for use with the compositions and
methods of treatment described herein include, but are not limited
to alkylating agents such as thiotepa and cyclosphosphamide; alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(especially bullatacin and bullatacinone); a camptothecin
(including the synthetic analogue topotecan); bryostatin;
callystatin; CC-1065 (including its adozelesin, carzelesin and
bizelesin synthetic analogues); cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gammalI and calicheamicin omegalI; dynemicin, including dynemicin
A; bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne
antiobiotic chromophores, aclacinomysins, actinomycin,
authrarnycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and
doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;
mercaptopurine; methotrexate; platinum coordination complexes such
as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;
vinorelbine; novantrone; teniposide; edatrexate; daunomycin;
aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);
topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);
retinoids such as retinoic acid; capecitabine; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0099] In another embodiment, the composition of the invention may
comprise other biologically active substances, including
therapeutic drugs or pro-drugs, for example, other chemotherapeutic
agents, scavenger compounds, antibiotics, anti-virals,
anti-fungals, anti-inflammatories, vasoconstrictors and
anticoagulants, antigens useful for cancer vaccine applications or
corresponding pro-drugs.
[0100] Exemplary scavenger compounds include, but are not limited
to thiol-containing compounds such as glutathione, thiourea, and
cysteine; alcohols such as mannitol, substituted phenols; quinones,
substituted phenols, aryl amines and nitro compounds.
[0101] Various forms of the chemotherapeutic agents and/or other
biologically active agents may be used. These include, without
limitation, such forms as uncharged molecules, molecular complexes,
salts, ethers, esters, amides, and the like, which are biologically
active.
E. Methods of Making Cyclodextrin/ATP Inhibitor Compositions
[0102] Methods of preparing cyclodextrin/ATP inhibitor compositions
and formulations thereof include the step of bringing into
association a compound of the present invention with the carrier
and, optionally, one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association a selective inhibitor of ATP production described
herein with a cyclodextrin. Generally, such complexes can be
obtained by agitating and mixing the cyclodextrin (e.g., a solution
containing the cyclodextrin) upon dropwise addition of the
therapeutic agent (e.g., a solution containing the selective
inhibitor of ATP production) or vice versa. Many mixing means are
known in the art to aid in combining the inhibitor and cyclodextrin
for example, without limitation, sonication, vortexing, stirring,
heating, co-precipitation, neutralization, slurrying, kneading,
grinding, and the like. It is possible to use a substance dissolved
in a solvent or a solid substance as the therapeutic agent
according to the physical properties of the therapeutic agent.
There are no particular limitations on the solvent, and one can
use, for example, a substance identical to the cyclodextrin
external phase. The amount of the therapeutic agent that is mixed
with the cyclodextrin can be equimolar quantities or in different
ratios depending on the desired level of incorporation. In some
embodiments, absolute amounts of the selective inhibitor of ATP
production can range between 0.001 to 10 mol equivalents, 0.01 to 1
mol equivalent, or any range inclusive relative to the amount of
cyclodextrin. Also, there are no particular limitations on the
heating temperature. For example, 5.degree. C. or higher, room
temperature or higher (e.g., 20.degree. C. or higher is also
preferable), are all acceptable.
[0103] Well-known methods exist for removing any undesired or
unincorporated complexes or compositions, such as therapeutic agent
not encapsulated by cyclodextrins or therapeutic agent cyclodextrin
complexes not encapsulated by liposomes. Representative examples
include, without limitation, dialysis, centrifugal separation, and
gel filtration. Dialysis can be conducted, for example, using a
dialysis membrane. As a dialysis membrane, one may cite a membrane
with molecular weight cut-off such as a cellulose tube or
Spectra/Por. With respect to centrifugal separation, centrifugal
acceleration any be conducted preferably at 100,000 g or higher,
and more preferably at 300,000 g or higher. Gel filtration may be
carried out, for example, by conducting fractionation based on
molecular weight using a column such as Sephadex or Sepharose.
[0104] In some cases, in order to prolong the effect of a drug, it
is desirable to modify (e.g., slow) the absorption of the drug from
subcutaneous or intramuscular injection. This may be accomplished
by the use of a liquid suspension of crystalline or amorphous
material having poor water solubility. The rate of absorption of
the drug then depends upon its rate of dissolution which, in turn,
may depend upon crystal size and crystalline form. Alternatively,
delayed absorption of a parenterally-administered drug form can be
accomplished by dissolving or suspending the drug in an oil
vehicle. In some embodiments, the cyclodextrin-encapsulated
selective ATP inhibitor compositions described herein can be loaded
into liposomes.
[0105] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
F. Therapeutic Methods
[0106] The present invention further provides novel therapeutic
methods of preventing, delaying, reducing, and/or treating a
cancer, including a cancerous tumor. In one embodiment, a method of
treatment comprises administering to a subject (e.g., a subject in
need thereof), an effective amount of a cyclodextrin/selective ATP
production inhibitor composition. A subject in need thereof may
include, for example, a subject who has been diagnosed with a
tumor, including a pre-cancerous tumor, a cancer, or a subject who
has been treated, including subjects that have been refractory to
the previous treatment.
[0107] The term "effective amount," as in "a therapeutically
effective amount," of a therapeutic agent refers to the amount of
the agent necessary to elicit the desired biological response. As
will be appreciated by those of ordinary skill in this art, the
effective amount of an agent may vary depending on such factors as
the desired biological endpoint, the agent to be delivered, the
composition of the pharmaceutical composition, the target tissue or
cell, and the like. More particularly, the term "effective amount"
refers to an amount sufficient to produce the desired effect, e.g.,
to reduce or ameliorate the severity, duration, progression, or
onset of a disease, disorder, or condition, or one or more symptoms
thereof; prevent the advancement of a disease, disorder, or
condition, cause the regression of a disease, disorder, or
condition; prevent the recurrence, development, onset or
progression of a symptom associated with a disease, disorder, or
condition, or enhance or improve the prophylactic or therapeutic
effect(s) of another therapy.
[0108] The methods of the present invention may be used to treat
any cancerous or pre-cancerous tumor. In certain embodiments, the
cancerous tumor has a highly glycolytic phenotype. For example,
highly glycolytic tumors may be located in a tissue selected from
brain, colon, urogenital, lung, renal, prostate, pancreas, liver,
esophagus, stomach, hematopoietic, breast, thymus, testis, ovarian,
skin, bone marrow and/or uterine tissue. In some embodiments,
methods and compositions of the present invention may be used to
treat any cancer. Cancers that may treated by methods and
compositions of the invention include, but are not limited to,
cancer cells from the bladder, blood, bone, bone marrow, brain,
breast, colon, esophagus, gastrointestine, gum, head, kidney,
liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach,
testis, tongue, or uterus. In addition, the cancer may specifically
be of the following histological type, though it is not limited to
these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated;
giant and spindle cell carcinoma; small cell carcinoma; papillary
carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma;
basal cell carcinoma; pilomatrix carcinoma; transitional cell
carcinoma; papillary transitional cell carcinoma; adenocarcinoma;
gastrinoma, malignant; cholangiocarcinoma; hepatocellular
carcinoma; combined hepatocellular carcinoma and
cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic
carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,
familial polyposis coli; solid carcinoma; carcinoid tumor,
malignant; branchiolo-alveolar adenocarcinoma; papillary
adenocarcinoma; chromophobe carcinoma; acidophil carcinoma;
oxyphilic adenocarcinoma; basophil carcinoma; clear cell
adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;
papillary and follicular adenocarcinoma; nonencapsulating
sclerosing carcinoma; adrenal cortical carcinoma; endometroid
carcinoma; skin appendage carcinoma; apocrine adenocarcinoma;
sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid
carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma;
papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma;
mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating
duct carcinoma; medullary carcinoma; lobular carcinoma;
inflammatory carcinoma; paget's disease, mammary; acinar cell
carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous
metaplasia; thymoma, malignant; ovarian stromal tumor, malignant;
thecoma, malignant; granulosa cell tumor, malignant; and
roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor,
malignant; lipid cell tumor, malignant; paraganglioma, malignant;
extra-mammary paraganglioma, malignant; pheochromocytoma;
glomangiosarcoma; malignant melanoma; amelanotic melanoma;
superficial spreading melanoma; malig melanoma in giant pigmented
nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma;
fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma;
liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal
rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed
tumor, malignant; mullerian mixed tumor; nephroblastoma;
hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner
tumor, malignant; phyllodes tumor, malignant; synovial sarcoma;
mesothelioma, malignant; dysgerminoma; embryonal carcinoma;
teratoma, malignant; struma ovarii, malignant; choriocarcinoma;
mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma,
malignant; kaposi's sarcoma; hemangiopericytoma, malignant;
lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;
chondrosarcoma; chondroblastoma, malignant; mesenchymal
chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;
odontogenic tumor, malignant; ameloblastic odontosarcoma;
ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma,
malignant; chordoma; glioma, malignant; ependymoma; astrocytoma;
protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma;
glioblastoma; oligodendroglioma; oligodendroblastoma; primitive
neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma;
neuroblastoma; retinoblastoma; olfactory neurogenic tumor;
meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant;
granular cell tumor, malignant; malignant lymphoma; Hodgkin's
disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma,
small lymphocytic; malignant lymphoma, large cell, diffuse;
malignant lymphoma, follicular; mycosis fungoides; other specified
non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma;
mast cell sarcoma; immunoproliferative small intestinal disease;
leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia;
lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia;
eosinophilic leukemia; monocytic leukemia; mast cell leukemia;
megakaryoblastic leukemia; myeloid sarcoma; and hairy cell
leukemia.
[0109] The compositions described herein may be delivered by any
suitable route of administration, including orally, nasally,
transmucosally, ocularly, rectally, intravaginally, parenterally,
including intramuscular, subcutaneous, intramedullary injections,
as well as intrathecal, direct intraventricular, intravenous,
intra-articular, intra-sternal, intra-synovial, intra-hepatic,
intralesional, intracranial, intraperitoneal, intranasal, or
intraocular injections, intracisternally, topically, as by powders,
ointments or drops (including eyedrops), including buccally and
sublingually, transdermally, through an inhalation spray, or other
modes of delivery known in the art.
[0110] The terms "systemic administration," "administered
systemically," "peripheral administration," and "administered
peripherally" as used herein mean the administration of the
selective ATP production inhibitor/cyclodextrin complex such that
it enters the patient's system and, thus, is subject to metabolism
and other like processes.
[0111] The terms "parenteral administration" and "administered
parenterally" as used herein mean modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intarterial, intrathecal, intracapsular, intraorbital, intraocular,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0112] In certain embodiments the pharmaceutical compositions are
delivered generally (e.g., via oral or parenteral administration).
In certain other embodiments the pharmaceutical compositions are
delivered locally through direct injection into a tumor or direct
injection into the tumor's blood supply (e.g., arterial or venous
blood supply). In some embodiments, the pharmaceutical compositions
are delivered by both a general and a local administration. For
example, a subject with a tumor may be treated through direct
injection of a composition containing a composition described
herein into the tumor or the tumor's blood supply in combination
with oral administration of a pharmaceutical composition of the
present invention. If both local and general administration is
used, local administration can occur before, concurrently with
and/or after general administration.
[0113] In certain embodiments, the methods of treatment of the
present invention, including treating a cancerous or pre-cancerous
tumor comprise administering compositions described herein in
combination with a second agent and/or therapy to the subject. By
"in combination with" is meant the administration of the selective
ATP production inhibitor/cyclodextrin complexes with one or more
therapeutic agents either simultaneously, sequentially, or a
combination thereof. Therefore, a subject administered a
combination of the selective ATP production inhibitor/cyclodextrin
complexes and/or therapeutic agents, can receive the selective ATP
production inhibitor/cyclodextrin complexes as described herein,
and one or more therapeutic agents at the same time (i.e.,
simultaneously) or at different times (i.e., sequentially, in
either order, on the same day or on different days), so long as the
effect of the combination of both agents is achieved in the
subject. When administered sequentially, the agents can be
administered within 1, 5, 10, 30, 60, 120, 180, 240 mins. or longer
of one another. In other embodiments, agents administered
sequentially, can be administered within 1, 5, 10, 15, 20 or more
days of one another.
[0114] When administered in combination, the effective
concentration of each of the agents to elicit a particular
biological response may be less than the effective concentration of
each agent when administered alone, thereby allowing a reduction in
the dose of one or more of the agents relative to the dose that
would be needed if the agent was administered as a single agent.
The effects of multiple agents may, but need not be, additive or
synergistic. The agents may be administered multiple times. In such
combination therapies, the therapeutic effect of the first
administered agent is not diminished by the sequential,
simultaneous or separate administration of the subsequent
agent(s).
[0115] Such methods in certain embodiments comprise administering
pharmaceutical compositions comprising compositions described
herein in conjunction with one or more chemotherapeutic agents
and/or scavenger compounds, including chemotherapeutic agents
described herein, as well as other agents known in the art.
Conjunctive therapy includes sequential, simultaneous and separate,
or co-administration of the composition in a way that the
therapeutic effects of the first selective ATP inhibitor
administered have not entirely disappeared when the subsequent
compound is administered. In one embodiment, the second agent is a
chemotherapeutic agent. In another embodiment, the second agent is
a scavenger compound. In another embodiment, the second agent is
radiation therapy. In a further embodiment, radiation therapy may
be administered in addition to the composition. In certain
embodiments, the second agent may be co-formulated in the separate
pharmaceutical composition.
[0116] In some embodiments, the subject pharmaceutical compositions
of the present invention will incorporate the substance or
substances to be delivered in an amount sufficient to deliver to a
patient a therapeutically effective amount of an incorporated
therapeutic agent or other material as part of a prophylactic or
therapeutic treatment. The desired concentration of the active
compound in the particle will depend on absorption, inactivation,
and excretion rates of the drug as well as the delivery rate of the
compound. It is to be noted that dosage values may also vary with
the severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions. Typically,
dosing will be determined using techniques known to one skilled in
the art.
[0117] Dosage may be based on the amount of the composition or
active compound thereof (e.g., selective inhibitor of ATP
production) per kg body weight of the patient. For example, a range
of amounts of compositions or compound encapsulated therein are
contemplated, including about 0.001, 0.01, 0.1, 0.5, 1, 10, 15, 20,
25, 50, 75, 100, 150, 200 or 250 mg or more of such compositions
per kg body weight of the patient. Other amounts will be known to
those of skill in the art and readily determined.
[0118] In certain embodiments, the dosage of the composition or
active compound thereof (e.g., selective inhibitor of ATP
production) will generally be in the range of about 0.001 mg to
about 250 mg per kg body weight, specifically in the range of about
50 mg to about 200 mg per kg, and more specifically in the range of
about 100 mg to about 200 mg per kg. In one embodiment, the dosage
is in the range of about 150 mg to about 250 mg per kg. In another
embodiment, the dosage is about 200 mg per kg.
[0119] In some embodiments the molar concentration of the
composition or active compound thereof (e.g., selective inhibitor
of ATP production) in a pharmaceutical composition will be less
than or equal to about 2.5 M, 2.4 M, 2.3 M, 2.2 M, 2.1 M, 2 M, 1.9
M, 1.8 M, 1.7 M, 1.6 M, 1.5 M, 1.4 M, 1.3 M, 1.2 M, 1.1 M, 1 M, 0.9
M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M or 0.2 M. In some
embodiments the concentration of the composition or active compound
thereof (e.g., selective inhibitor of ATP production) will be less
than or equal to about 0.10 mg/ml, 0.09 mg/ml, 0.08 mg/ml, 0.07
mg/ml, 0.06 mg/ml, 0.05 mg/ml, 0.04 mg/ml, 0.03 mg/ml or 0.02
mg/ml.
[0120] Alternatively, the dosage may be determined by reference to
the plasma concentrations of the composition or active compound
thereof (e.g., selective inhibitor of ATP production). For example,
the maximum plasma concentration (C.sub.max) and the area under the
plasma concentration-time curve from time 0 to infinity (AUC (0-4))
may be used. Dosages for the present invention include those that
produce the above values for C.sub.max and AUC (0-4) and other
dosages resulting in larger or smaller values for those
parameters.
[0121] Actual dosage levels of the active ingredients in the
compositions of the present invention may be varied so as to obtain
an amount of the active ingredient which is effective to achieve
the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0122] The selected dosage level will depend upon a variety of
factors including the activity of the particular therapeutic agent
in the formulation employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion or metabolism of the particular therapeutic agent
being employed, the duration of the treatment, other drugs,
compounds and/or materials used in combination with the particular
compound employed, the age, sex, weight, condition, general health
and prior medical history of the patient being treated, and like
factors well known in the medical arts.
[0123] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could prescribe and/or administer doses of the
compounds of the invention employed in the pharmaceutical
composition at levels lower than that required in order to achieve
the desired therapeutic effect and gradually increase the dosage
until the desired effect is achieved.
[0124] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above.
[0125] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms.
[0126] The precise time of administration and amount of any
particular compound that will yield the most effective treatment in
a given patient will depend upon the activity, pharmacokinetics,
and bioavailability of a particular compound, physiological
condition of the patient (including age, sex, disease type and
stage, general physical condition, responsiveness to a given dosage
and type of medication), route of administration, and the like. The
guidelines presented herein may be used to optimize the treatment,
e.g., determining the optimum time and/or amount of administration,
which will require no more than routine experimentation consisting
of monitoring the subject and adjusting the dosage and/or
timing.
[0127] While the subject is being treated, the health of the
patient may be monitored by measuring one or more of the relevant
indices at predetermined times during a 24-hour period. All aspects
of the treatment, including supplements, amounts, times of
administration and formulation, may be optimized according to the
results of such monitoring. The patient may be periodically
reevaluated to determine the extent of improvement by measuring the
same parameters, the first such reevaluation typically occurring at
the end of four weeks from the onset of therapy, and subsequent
reevaluations occurring every four to eight weeks during therapy
and then every three months thereafter. Therapy may continue for
several months or even years, with a minimum of one month being a
typical length of therapy for humans. Adjustments, for example, to
the amount(s) of agent administered and to the time of
administration may be made based on these reevaluations.
[0128] Treatment may be initiated with smaller dosages which are
less than the optimum dose of the compound. Thereafter, the dosage
may be increased by small increments until the optimum therapeutic
effect is attained.
[0129] As described above, the composition or active compound
thereof (e.g., selective inhibitor of ATP production) may be
administered in combination with radiation therapy. An optimized
dose of radiation therapy may be given to a subject as a daily
dose. Optimized daily doses of radiation therapy may be, for
example, from about 0.25 to 0.5 Gy, about 0.5 to 1.0 Gy, about 1.0
to 1.5 Gy, about 1.5 to 2.0 Gy, about 2.0 to 2.5 Gy, and about 2.5
to 3.0 Gy. An exemplary daily dose may be, for example, from about
2.0 to 3.0 Gy. A higher dose of radiation may be administered, for
example, if a tumor is resistant to lower doses of radiation. High
doses of radiation may reach, for example, 4 Gy. Further, the total
dose of radiation administered over the course of treatment may,
for example, range from about 50 to 200 Gy. In an exemplary
embodiment, the total dose of radiation administered over the
course of treatment ranges, for example, from about 50 to 80 Gy. In
certain embodiments, a dose of radiation may be given over a time
interval of, for example, 1, 2, 3, 4, or 5 mins., wherein the
amount of time is dependent on the dose rate of the radiation
source.
[0130] In certain embodiments, a daily dose of optimized radiation
may be administered, for example, 4 or 5 days a week, for
approximately 4 to 8 weeks. In an alternate embodiment, a daily
dose of optimized radiation may be administered daily seven days a
week, for approximately 4 to 8 weeks. In certain embodiments, a
daily dose of radiation may be given a single dose. Alternately, a
daily dose of radiation may be given as a plurality of doses. In a
further embodiment, the optimized dose of radiation may be a higher
dose of radiation than can be tolerated by the patient on a daily
base. As such, high doses of radiation may be administered to a
patient, but in a less frequent dosing regimen.
[0131] The types of radiation that may be used in cancer treatment
are well known in the art and include electron beams, high-energy
photons from a linear accelerator or from radioactive sources such
as cobalt or cesium, protons, and neutrons. An exemplary ionizing
radiation is an x-ray radiation.
[0132] Methods of administering radiation are well known in the
art. Exemplary methods include, but are not limited to, external
beam radiation, internal beam radiation, and radiopharmaceuticals.
In external beam radiation, a linear accelerator is used to deliver
high-energy x-rays to the area of the body affected by cancer.
Since the source of radiation originates outside of the body,
external beam radiation can be used to treat large areas of the
body with a uniform dose of radiation. Internal radiation therapy,
also known as brachytherapy, involves delivery of a high dose of
radiation to a specific site in the body. The two main types of
internal radiation therapy include interstitial radiation, wherein
a source of radiation is placed in the effected tissue, and
intracavity radiation, wherein the source of radiation is placed in
an internal body cavity a short distance from the affected area.
Radioactive material may also be delivered to tumor cells by
attachment to tumor-specific antibodies. The radioactive material
used in internal radiation therapy is typically contained in a
small capsule, pellet, wire, tube, or implant. In contrast,
radiopharmaceuticals are unsealed sources of radiation that may be
given orally, intravenously or directly into a body cavity.
[0133] Radiation therapy may also include stereotactic surgery or
stereotactic radiation therapy, wherein a precise amount of
radiation can be delivered to a small tumor area using a linear
accelerator or gamma knife and three dimensional conformal
radiation therapy (3DCRT), which is a computer assisted therapy to
map the location of the tumor prior to radiation treatment.
[0134] Toxicity and therapeutic efficacy of subject compounds may
be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., for determining the
LD.sub.50 and the ED.sub.50. Compositions that exhibit large
therapeutic indices are preferred. In some embodiments, the
LD.sub.50 (lethal dosage) can be measured and can be, for example,
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,
300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more reduced for
the cyclodextrin-encapsulated selective ATP inhibitor compositions
described herein relative to the selective ATP inhibitor without
any cyclodextrin encapsulation. Similarly, the ED.sub.50 (i.e., the
concentration which achieves a half-maximal inhibition of symptoms)
can be measured and can be, for example, at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%,
700%, 800%, 900%, 1000% or more increased for the
cyclodextrin-encapsulated selective ATP inhibitor compositions
described herein relative to the selective ATP inhibitor without
any cyclodextrin encapsulation. Also, Similarly, the IC.sub.50
(i.e., the concentration which achieves half-maximal cytotoxic or
cytostatic effect on cancer cells) can be measured and can be, for
example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more
increased for the cyclodextrin-encapsulated selective ATP inhibitor
compositions described herein relative to the selective ATP
inhibitor without any cyclodextrin encapsulation. Although
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets the compounds to
the desired site in order to reduce side effects.
[0135] In some embodiments, the presently disclosed methods produce
at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% inhibition of
cancer cell growth in an assay.
[0136] In any of the above-described methods, the administering of
the selective ATP production inhibitor/cyclodextrin complexes can
result in at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%
decrease in a solid malignancy in a subject, compared to the solid
malignancy before administration of the selective ATP production
inhibitor/cyclodextrin complexes.
[0137] In some embodiments, the therapeutically effective amount of
a complex of a selective ATP production inhibitor/cyclodextrin is
administered prophylactically to prevent a solid malignancy from
forming in the subject.
[0138] In some embodiments, the subject is human. In other
embodiments, the subject is non-human, such as a mammal.
[0139] The data obtained from the cell culture assays and animal
studies may be used in formulating a range of dosage for use in
humans. The dosage of any supplement, or alternatively of any
components therein, lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. For
agents of the present invention, the therapeutically effective dose
may be estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range that includes the IC.sub.50 as determined in
cell culture. Such information may be used to more accurately
determine useful doses in humans. Levels in plasma may be measured,
for example, by high performance liquid chromatography.
G. Kit
[0140] The selective ATP production inhibitor/cyclodextrin
complexes and compositions described herein can be assembled into
kits or pharmaceutical systems for use in treating or preventing a
disease, such as cancer. In some embodiments, the
3-BrPA-cyclodextrin complex and compositions can be used to prevent
or treat solid malignancies caused by a cancer. In general, a
presently disclosed kit contains some or all of the components,
reagents, supplies, and the like to practice a method according to
the presently disclosed subject matter. The kit typically comprises
an effective amount of complex to prevent, delay, reduce, or treat
an unwanted disease (e.g., a solid malignancy). In one embodiment,
a kit comprises at least one container (e.g., a carton, bottle,
vial, tube, or ampoule) comprising a selective ATP production
inhibitor/cyclodextrin complex and/or compositions thereof
described herein. Typically, the complex and/or compositions will
be supplied in one or more container, each container containing an
effective amount of complex to allow a solid malignancy to regress,
slow, or be arrested.
[0141] Accordingly, in some embodiments, the presently disclosed
subject matter provides a kit comprising at least one selective ATP
production inhibitor encapsulated within at least one cyclodextrin
carrier. In other embodiments, the kit further comprises a set of
instructions for using the at least one selective ATP production
inhibitor encapsulated within the at least one cyclodextrin
carrier.
[0142] It may be desirable to store the selective ATP production
inhibitor and cyclodextrin separately and then combine them before
use. Accordingly, in still other embodiments, the kit comprises at
least one selective ATP production inhibitor in one container and
at least one cyclodextrin carrier in another container.
EXEMPLIFICATION
[0143] The following Examples have been included to provide
guidance to one of ordinary skill in the art for practicing
representative embodiments of the presently disclosed subject
matter. In light of the present disclosure and the general level of
skill in the art, those of skill can appreciate that the following
Examples are intended to be exemplary only and that numerous
changes, modifications, and alterations can be employed without
departing from the scope of the presently disclosed subject matter.
The following Examples are offered by way of illustration and not
by way of limitation.
Example 1: Materials and Methods for Examples 2-3
[0144] A. General method for synthesis of modified
.beta.-cyclodextrins
[0145] Succinyl-.beta.-cyclodextrins were purchased from Sigma
Chemical (St. Louis, Mo., USA; Catalog No. 85990). Unmodified
.beta.-cyclodextrin and .alpha.-cyclodextrin were purchased
(Sigma-Aldrich, St. Louis, Mo.).
[0146] However, succinylated cyclodextrins can also be synthesized.
For example, .beta.-cyclodextrin (Sigma-Aldrich, St. Louis, Mo.)
was mono-tosylated with 0.9 molar equivalent of tosyl chloride in
pyridine at the primary 6' hydroxyl group to afford the
corresponding tosylate, which was converted to the iodo-derivative
by treatment with sodium iodide in acetone. The iodo derivative was
converted to the desired 6' aminated cyclodextrin by heating at
80.degree. C. for 8-12 hours with the appropriate amine (Tang and
Ng (2008) Nat. Protocol. 3:691-697). 6'
mono-succinyl-.beta.-cyclodextrin was synthesized by treatment of
parent .beta.-cyclodextrin with 0.9 equivalents of succinic
anhydride in DMF (Cucinotta et al. (2005) J. Pharmaceut. Biomed.
Anal. 37:1009-1014). The product was precipitated in acetone and
purified by HPLC before use.
[0147] The pH range with optimal stability is pH 4-9.
[0148] B. General procedure of preparation of encapsulated
complexes
[0149] A 1:1 ratio of 3-BrPA encapsulated within
succinyl-.beta.-cyclodextrins was prepared. 3-BrPA (150 mg, 1 mmol)
was added in small portions (10 mg each) to a stirring solution of
succinyl-beta-cyclodextrin (1,500 mg in distilled water). After
complete addition, the solution was sonicated for 1 hour at room
temperature. The sonicated solution was then allowed to shake
overnight on a thermomixer at 25.degree. C., flash frozen in a dry
ice-acetone bath, and lyophilized.
[0150] Similarly, a 2:1 ratio of 3-BrPA encapsulated within
succinyl-.beta.-cyclodextrins was prepared. 3-BrPA (166 mg, 1 mmol)
was added in small portions (10 mg each) to a stirring solution of
succinyl-beta-cyclodextrin (918 mg in 20 ml distilled water). After
complete addition, the solution was sonicated for 1 hour at room
temperature. The sonicated solution was then allowed to shake
overnight on a thermomixer at 25.degree. C., flash frozen in a dry
ice-acetone bath, and lyophilized.
[0151] In addition, a 1:1 ratio of 3-BrPA encapsulated within
.alpha.-cyclodextrins (see structure below) was prepared. 3-BrPA
(166 mg, 1 mmol) was added in small portions (10 mg each) to a
stirring solution of alpha-cyclodextrin (972 mg, 1 mmol in 10 ml
distilled water). After complete addition, the solution was
sonicated for 1 hour at room temperature. The sonicated solution
was then allowed to shake overnight on a thermomixer at 25.degree.
C., flash frozen in a dry ice-acetone bath, and lyophilized.
Non-GRAS and GRAS versions were used with similar results.
##STR00003##
[0152] It was surprisingly determined that cyclodextrins modified
to replace one or more hydroxyl groups on one or more of its
.alpha.-D-glucopyranoside units with ionizable groups resulting in
negative charges (anions) stabilizes the 3-halopyruvates better
than those having ionizable groups resulting in positive charges
(cations) or unmodified cyclodextrins, such as unmodified alpha- or
beta-cyclodextrin. It was also surprisingly determined that
.beta.-cyclodextrins encapsulate 3-BrPA in a form that protects and
stabilizes 3-BrPA for in vivo efficacy especially and also in vitro
efficacy significantly better than .alpha.-cyclodextrins.
[0153] In addition, in vitro cell culture and in vivo mouse
treatments for were prepared and performed as described above and
below for succinyl-.beta.-cyclodextrins encapsulating 3-BrPA. using
generally recognized as safe (GRAS) versions of
.beta.-cyclodextrins (e.g., hydroxypropyl-.beta.-cyclodextrin
having a level of substitution of 3-5 such as that shown in
chemical form below) encapsulating 3-BrPA and the results were
similar to those described for succinyl-fl-cyclodextrins
encapsulating 3-BrPA.
##STR00004##
[0154] C. In Vitro Cell Culture
[0155] 3-BrPA and .beta.-cyclodextrin (vehicle) were purchased from
Sigma Chemical (St. Louis, Mo., USA). For the viability assay,
MiaPaCa-2 and Suit-2 cells were seeded in triplicates in 96-well
plates at a density of 5.times.10.sup.3 cells per well. After 12
hours, cells were treated with increasing concentrations of 3-BrPA,
CD-3BrPA (0-150 .mu.m) and the vehicle. Intracellular ATP levels
were measured using a Cell Titer-Glo Luminescence Cell Viability
assay kit (Promega, Durham, N.C., USA) according to the
manufacturer's protocol. The measurements were performed at 24
hours and 72 hours after the treatment.
[0156] D. In Vivo Mouse Treatment
[0157] A total of 15 animals were randomized to receive daily
injections with 5 mg/kg Beta-CD-3BrPA (in a 1:1 ratio) (N=10) or
vehicle control (N=5). Baseline bioluminescence imaging confirmed
tumor growth in all animals (five representative animals shown in
FIG. 4 and FIG. 5). After two weeks of intra-peritoneal injections,
all animals were subjected to follow-up imaging. Animals treated
with the vehicle demonstrated a strong increase of the
bioluminescence signal, representing tumor progression.
[0158] Male athymic nude mice (20-25 g, 4 weeks old, Crl:Nu-Nu,
Charles River Laboratories, Wilmington, Mass., USA) were used in
accordance with the institutional guidelines under approved
protocols. Mice were maintained in laminar flow rooms at constant
temperature and humidity with food and water given ad libitum. The
MiaPaCa-2 cell line, stably transfected with the
luciferase-aminoglycoside phosphotransferase fusion gene under the
control of the elongation factor 1 alpha promoter was used. Mice
were anesthetized by isoflurane inhalation anesthesia before
surgery and treatment. A small left abdominal flank incision was
made and the pancreas was exteriorized. Orthotopic pancreatic
tumors were generated by injection of 1-2.times.10.sup.6 MiaPaCa-2
cells into the tail of the pancreas. A successful subcapsular
intrapancreatic injection of the tumor cells was identified by the
appearance of a fluid bleb without intraperitoneal leakage.
[0159] For bioluminescence imaging (BLI), anesthetized mice bearing
orthotopic tumors were injected intraperitoneally with 150 mg/kg of
D-Luciferin (Gold Biotechnilogy, St Louis, Mo., USA) and optically
imaged after 5 mins. using the IVIS 100 (Xenogen Corp, Alameda,
Calif., USA). The pseudocolor image which represented the spatial
distribution of detected photons was overlaid on a grayscale
photographic image. Signal intensity was quantified with ROIs
(p/s/cm.sup.2/Sr) after a 10-second exposure using Living Image
software (Xenogen Corp.). Imaging was performed on day 7, 14, 21,
28 and 35 after tumor implantation.
[0160] Following the confirmation of tumor growth in each animal
using BLI one week after tumor implantation, all animals were
randomized in 3 groups to receive either 3-BrPA, CD-3BrPA or the
vehicle via daily intra-peritoneal injections (injection volume,
500 .mu.l/mouse/day; dose, 5 mg/kg). The injection solution was
prepared by dissolving the chemicals in phosphate buffered saline,
adjusted to a pH of 7.4. Animals were observed once per hour during
the initial injections and every 4-6 hours after every follow-up
injection. Any changes in the overall clinical condition were noted
for all treatment groups.
[0161] Within 24 hours after the last BLI imaging, animals were
sacrificed using cervical dislocation. The entire abdomen was
opened and tumors were obtained using en-bloc extraction with the
spleen and pancreas. Tumor specimens were fixed using 4%
paraformaldehyde for 72 hours, paraffin embedded, and sectioned.
Histological sections were hematoxylin and eosin (H&E) stained
and interpreted in consultation with a pathologist.
Example 2: In Vitro Effects of Cyclodextrins Encapsulating 3-BrPA
on Human Pancreatic Cancer Cell Lines
[0162] Two cell lines of human pancreatic cancer, namely MiapaCa-2
and Suit-2, were tested for their response to 3-BrPA and CD-3-BrPA.
MiaPaCa-2 is derived from a locally invasive human adenocarcinoma
and forms typical solid nodules within the pancreas. It is known to
show a pronounced resistance to several standard-of-care anticancer
agents, including gemcitabine. Suit-2 is derived from a highly
aggressive pancreatic tumor that has been isolated from a
metastatic liver mass. It has highly aggressive phenotypic
properties, such as invasion and migration. The cellular response
or the cell viability was assayed using the standard ATP viability
assay. Each experiment was repeated at least twice, with triplicate
biological samples.
[0163] FIGS. 2A-2B show the effects of 3-BrPA (conventional 3-BrPA
in phosphate-buffered saline without any cyclodextrin encapsulation
or complex formation) or beta-CD-3-BrPA
(succinyl-.beta.-cyclodextrins encapsulating 3-BrPA in
phosphate-buffered saline) on MiaPaCa-2 cells (FIG. 2A) and Suit-2
cells (FIG. 2B) after 24 hours of treatment. The data show a dose
dependent decrease in viability of cells treated with
beta-CD-3-BrPA compared to 3-BrPA treated cells. In addition, the
multi-drug resistant MiaPaCa2 cells were found to be more sensitive
to 3-BrPA/CD-3-BrPA than Suit2 cells. The vehicle
(.beta.-cyclodextrin in phosphate-buffered saline without any
3-BrPA) by itself did not contribute to any toxicity.
[0164] FIG. 3 shows the effects of 3-BrPA (conventional 3-BrPA in
phosphate-buffered saline without any cyclodextrin encapsulation or
complex formation) or beta-CD-3-BrPA (succinyl-.beta.-cyclodextrins
encapsulating BrPA in phosphate-buffered saline) on MiaPaCa2 cells
after 72 hours of treatment. MiaPaCa-2 cells show a significant
loss of viability even at .about.50 uM concentration of CD-3-BrPA
at 72 hours of treatment, whereas at the same concentration at 24
hours (FIG. 2A), there was no significant loss of viability. Thus,
with longer duration (48 hrs.) of treatment, CD-3-BrPA at fairly
low concentration (50 uM) is sufficient to initiate cell death
(.about.50% death) (FIG. 3).
Example 3: Materials and In Vivo Effects of Cyclodextrins
Encapsulating 3-BrPA in a Mouse Model of Human Pancreatic
Cancer
[0165] An athymic mouse model of human pancreatic cancer was used
for in vivo studies. The human pancreatic cancer cell line,
MiaPaca-2, stably expressing the luciferase gene, was
orthotopically implanted onto the pancreas. Tumor growth and
response were monitored by bioluminescence imaging.
[0166] Table 1 describes the clinical signs or symptoms observed in
tumor-bearing animals treated with a high-dose of 3-BrPA or
CD-3-BrPA. These symptoms were recorded in an unbiased and
blind-study fashion. These symptoms were observed at a dose of
>5 mg/kg body weight and at a concentration of .about.3.5 mM
(higher than the recommended therapeutic dose). Undesirable
clinical signs or symptoms were seen in the mice treated with
3-BrPA and these signs or symptoms were significantly less in the
mice treated with CD-3-BrPA, indicating that the cyclodextrin
carrier protects the subject from the side effects of the 3-BrPA
molecule.
TABLE-US-00001 TABLE 1 Succinyl-.beta.- cyclodextrins-3-BrPA
Clinical Signs/Conditions 3-BrPA treatment treatment
Seizures/Shiver/Spasms +++ +/- Salivation +++ +/- Shortness of
Breath +++ +/- Abnormal Behavior (e.g., +++ +/-
inactive/unresponsive) Hypothermia +++ +/-
[0167] Animals treated with succinyl-.beta.-cyclodextrin-3-BrPA
showed complete or almost-complete tumor response on
bioluminescence imaging (FIG. 4 and FIG. 5). Subsequently, animals
were sacrificed to confirm the bioluminescence results with
necropsy (FIG. 4). The results indicate that CD-3BrPA preserves its
anticancer activity even after complex formation with CD.
[0168] Histopathological analysis of the orthotopic MiaPaCa-2
tumors was performed. FIG. 6 shows that hematoxylin and eosin
(H&E)-stained tumors showed no changes in the control group,
while tumors harvested from treated animals show extensive central
necrosis as well as areas of dissociating tumor tissue.
[0169] Thus, it has been determined that cyclodextrin complex
formation does not affect the anticancer properties of 3-BrPA, as
evident from both in vitro and in vivo data. Also, the activity of
3-BrPA can be preserved or protected by CD until it is delivered or
distributed to the target organ or tumor. In addition, CD-3-BrPA
administration to animals results in lesser toxicity or
related-clinical signs compared to 3-BrPA alone.
Example 4: Materials and Methods for Examples 5-6
[0170] The experiments described in Examples 2-3 were expanded in
order to advance the results obtained therefrom using the following
materials and methods. For example, pancreatic ductal
adenocarcinomas (PDAC) rank as the fourth most common cause for
cancer related death in the world (Siegel et al. (2014) CA Canc. J.
Clin. 64:9-29). As the majority of patients are diagnosed at
advanced stages, therapeutic options remain limited and the
prognosis is dismal with a 5-year survival rate of less than 5%
(Hidalgo (2010) New Engl. J. Med. 362:1605-1617). The last two
decades brought significant advances in the understanding of
tumorigenesis and disease progression in pancreatic cancer, which
can now be seen as a diverse and multifactorial neoplastic process
(Hidalgo (2010) New Engl. J. Med. 362:1605-1617; Hanahan and
Weinberg (2011) Cell 144:646-674). Pancreatic tumor tissue is
composed of several distinctive, cellular and non-cellular elements
including a collagen-rich, poorly vascularized and highly hypoxic,
non-neoplastic stroma (Chu et al. (2007) J. Cell. Biochem.
101:887-907; Mahadevan and Von Hoff (207) Mol. Canc. Therapeut.
6:1186-1197). These characteristics are associated with profound
chemoresistance to the most commonly used systemically applicable
anti-cancer agents, such as gemcitabine (Muerkoster et al. (2004)
Cancer Res. 64:1331-1337; Yokoi and Fidler (2004) Clin. Canc. Res.
10:2299-2306). Notably, altered energy metabolism has been recently
added to the organizing principles of tumor microenvironment and
can now be seen as a "hallmark" of pancreatic cancer (Hanahan and
Weinberg (2011) Cell 144:646-674; Guillaumond et al. (2014) Arch.
Biochem. Biophys 545:69-73. The oxygen-independent reliance on
glycolysis as the main axis of energy supply for cancer cells has
long been known as the "Warburg effect"; however, this circumstance
has not yet been clinically exploited for therapeutic purposes
(Warburg et al. (1927) J. Gen. Physiol. 8:519-530;
Ganapathy-Kanniappan and Geschwind (2013) Mol. Cancer 12:152).
3-bromopyruvate (3-BrPA), a highly potent small-molecular inhibitor
of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is
the only available anti-glycolytic drug candidate that is able to
enter cancer cells selectively through the monocarboxylate
transporter 1 (MCT1) (Ganapathy-Kanniappan et al. (2009) Anticancer
Res. 29:4909-4918; Birsoy et al. (2013) Nature Genet. 45:104-108).
The anti-tumoral effects of 3-BrPA have been extensively studied
and confirmed in several murine tumor models in the setting of
loco-regional therapy, delivered either through tumor-feeding
arteries or with direct intra-tumoral injections (Ota et al. (2013)
Target. Oncol. 8:145-151; Geschwind et al. (2002) Canc. Res.
62:3909-3913). However, due to its alkylating properties, 3-BrPA
has demonstrated significant toxicity when delivered systemically
in therapeutic doses, which in return could impede the clinical
development and use of this drug in cancer patients (Chang et al.
(2007) Acad. Radiol. 14:85-92; Cao et al. (2008) Clin. Canc. Res.
14:1831-1839).
[0171] A. Antibodies, Reagents, and Kits
[0172] The following primary antibodies were used: rabbit
anti-MMP-9 polyclonal antibody (pAB) #3852 (Cell Signaling), DAPI
#D1306 (Invitrogen), Alexa Fluor 568 Phalloidin #12380 (Life
Technologies), GAPDH (14C10) monoclonal AB (mAB) Alexa Fluor 488
Conjugate #3906 (Cell Signaling), GAPDH pAB #sc-47724 (Santa Cruz),
cleaved caspase-3 pAB #9661 (Cell Signaling), MCT-1 pAB #sc50324
(Santa Cruz), and a Ki-67 kit/antibody (Dako Inc.). The following
secondary antibodies were used: goat anti-rabbit IgG HRP-conjugated
#7074 (Santa Cruz), anti-rabbit IgG (H+L), F(ab').sub.2 fragment PE
conjugate #8885 (Cell Signaling), and goat anti-mouse IgG-FITC
#sc2010. The following chemicals were used: 3-bromopyruvatic acid
(3-BrPA, Sigma Aldrich), gemcitabine hydrochloride salt (LC
Laboratories), succinyl-.beta.-cyclodextrin (.beta.-CD, Sigma
Aldrich), and D-luciferin potassium salt (Gold Biotechnology, St
Louis, Mo., USA). The following cell culture reagents were used:
RPMI-1640 (Life Technologies), MEM (Life Technologies), fetal
bovine serum (FBS, Thermo Scientific), penicillin/streptomycin
(Sigma Aldrich), collagen I rat tail (BD Biosciences, #354326), and
controlled atmosphere chamber (Plas. Labs). The following invasion
assay reagents were used: matrigel basement membrane matrix (BD
Biosciences) and matrigel invasion chamber transwell polycarbonate
membrane inserts (Corning). The following kits were used:
CellTiter-Glo luminescence cell viability assay kit (Promega),
dual-luciferase reporter assay kit (Promega), 2D quant kit (GE
Healthcare), histostain plus 3rd gen ICH detection kit
(Invitrogen), and diff quik stain kit (Polysciences Inc.). The
following imaging equipment was used: Zeiss 700 LSM confocal
microscope, Olympus IX81 inverted microscope, Eclipse TS100
inverted microscope (Nikon), and IVIS200 (Xenogen Corp., Alameda,
Calif.)
[0173] B. Complex Preparation and Nuclear Magnetic Resonance (NMR)
Spectroscopy
[0174] To prepare 3-BrPA encapsulated in .beta.-CD, 3-BrPA (166 mg,
1 mM) was added in small portions (10 mg each) to a stirring
solution of .beta.-CD (918 mg in 20 ml DI water). After completing
the addition, the solution was sonicated for 1 hour at 50.degree.
C. The sonicated solution was then allowed to shake overnight on a
thermomixer at 25.degree. C., flash frozen in a dry ice-acetone
bath and lyophilized. The lyophilized complex was used as such for
further biological and biophysical studies. .sup.1H NMR experiments
were performed at 400 MHz on a Bruker Avance spectrometer. The NMR
spectra were recorded in 99.9% D20 and are reported in parts per
million downfield relative to tetramethysilance (TMS). Ten mM
solutions of .beta.-CD alone, 3-BrPA alone, or the complex of
3-BrPA and .beta.-CD, were prepared with 1% DSS
(3-(trimethylsilyl)-1-propanesulfonic acid, sodium salt; Sigma
Aldrich) as an internal standard. Spectra were recorded at
25.degree. C. with 32 scans. An upheld shift of the methylene
protons (0.1 ppm) was observed upon complexation (see FIG. 7).
[0175] C. Monolayer Cell Culture and Viability Assay
[0176] Two human pancreatic adenocarcinoma cell lines, lucMiaPaCa-2
(stably transfected with the luciferase-aminoglycoside
phosphotransferase fusion gene, kindly provided by Dr. Phuoc T.
Tran) and Suit-2 (kindly provided by Dr. Shinichi Ota, Japan) were
cultured in RPMI or MEM media, respectively, both supplemented with
10% FBS and 1% Penicillin-Streptomycin. The effects of different
drugs on cell viability were determined by quantifying
intracellular adenosine triphosphate (ATP) levels using a
luminescence-based kit (CellTiter-Glo, Promega) and a multilable
96-well plate (Costar). The accuracy and reproducibility of
viability measurements using this luminiscence-based kit in
lucMiaPaCa-2 cells was confirmed using the Dual-Reporter assay kit
(Promega). In brief, 5.times.10.sup.3 cells were seeded in
triplicate and incubated for 72 hrs. under normoxic or hypoxic (1%
O.sub.2-level, balanced with CO.sub.2 and nitrogen within a
controlled atmosphere chamber) conditions. Indicated amounts of
free 3-BrPA, 1:1-.beta.-CD-3-BrPA or .beta.-CD as a control were
dissolved in PBS and added to the medium for 24 hrs. of treatment.
For the experiments with gemcitabine, cells were incubated for 24
hrs. prior to a 72 hrs. exposure to the drug. Cell viability was
determined following the manufacturer's protocol.
[0177] D. 3D Organotypic Cell Culture, Imaging, and
Immunofluorescence
[0178] A collagen 1-based 3D organotypic cell culture was used to
mimick an extracellular-matrix (ECM)-rich environement and to test
the effects of 3-BrPA on tumor invasion (Cheung et al. (2013) Cell
155:1639-1651; Nguyen-Ngoc and Ewald (2013) J. Microscop.
251:212-223). Specifically, a collagen solution which initially
consisted of 25 .mu.l of 10.times. DMEM and 217 .mu.l of collagen I
(3.83 mg/ml) was prepared on ice. The pH value was adjusted by
dropwise addition of sodium hydroxide (Sigma Aldrich) to reach
pH=7.0. The collagen I was then diluted using DMEM F12/GlutaMAX
(Life Technologies) to a final concentration of 3 mg/ml. An
underlayer was created on the bottom of each well of an uncovered
glass-bottom 24-well plate (InVitroScientific) using 15 .mu.l of
the collagen solution, which was then allowed to polymerize at
37.degree. C. for at least 1 hr. The remaining collagen solution
was kept on ice for 3-5 hrs. to allow initial polymerization. A
total of 65.times.10.sup.3 lucMiaPaCa-2 or 45.times.10.sup.3 Suit-2
cells were resuspended in a volume of 150 .mu.l collagen solution.
By creating a drop with the height of 0.5 cm, the cell suspension
was placed on top of the pre-warmed underlayer. The collagen-cell
suspension was allowed to polymerize for 1 h at 37.degree. C. and
subsequently covered with cell culture medium (Nguyen-Ngoc and
Ewald (2013) J. Microscop. 251:212-223).
[0179] 3D organoids were treated either once or sequentially. For
single treatments, embedded cells were incubated for 5 days under
normoxic or hypoxic (1% O.sub.2-level within a controlled
atmosphere chamber) conditions prior to treatment. On day 5, medium
was replaced by 1:1-.beta.-CD-3-BrPA/3-BrPA/.beta.-CD-containing
medium and the cells were incubated for 24 hrs. with the respective
concentrations of the drug. For experiments with gemcitabine, cells
were allowed to grow for 48 hrs. before being treated and incubated
with the drug for another 72 hrs. Initial experiments with
gemcitabine did not demonstrate any efficacy after 24 hrs., and it
was decided to follow the most commonly reported incubation times
of 72 hrs. Sequential treatment with 3-BrPA was performed on
alternate days for one week with the respective doses and evaluated
by bright field microscopy (Olympus) at 40.times. magnification
with a 1.3 NA oil objective. A Hamamatsu Photonics C9100-02 EMCCD
camera was used to acquire the images with the SlideBook 5.0
program.
[0180] Microscopic observations were compared with the
quantification of cell viability as seen on in vitro
bioluminescence imaging (BLI). For the latter measurements, the
cell culture medium covering the 3D organotypic cell culture was
replaced by 500 ul of a luciferase substrate (D-luciferin,
potassium salt, Life Technologies, 20 mg/ml) in PBS. After 5 mins.
of exposure, the plate was positioned and images were acquired
(Xenogen Ivis Imaging System 100). Signal intensity was determined
by the photon emission (in counts) and measured within a region of
interest (ROI) which enclosed the entire 3D organoids (Living Image
Software, PerkinElmer).
[0181] The microscopic and BLI findings were verified using
immunofluorescence microscopy. 3D organoids were fixed using 4%
formaldehyde and cryofixed with OCT Compound (Tissue Tek) at
-80.degree. C. The samples were cut into sections of 100 .mu.m
thickness at -20.degree. C. OCT was washed off using PBS twice for
10 mins. Each. Prior to staining, sections were permeabilized with
0.5% Trizol 100 in PBS for 30 mins. and washed twice with PBS for
10 mins. each. After blocking with 10% FBS in PBS for 2 hrs.,
samples were incubated with primary antibodies (Alexa Fluor 568
Phalloidin, Invitrogen, 1:100; GAPDH Alexa Fluor 488 conjugate,
Cell Signalling, 1:800; cleaved caspase-3, Cell Signalling, 1:500;
HIF-1 alpha 1:50) for 1 hr. at room temperature (RT) under light
protection. For non-conjugated primary antibodies, additional
incubation with a phycoerythrin (PE)- or fluoresceine
isothiocyanate (FITC)-conjugated secondary antibody for 1 hr. at RT
was used. This was followed by two washings with PBS for 10 mins.
each. DAPI was used as a counter stain at a concentration of 300
ng/ml and added to the specimen simultaneously with the conjugated
antibody. Specimens were sealed with an antifading mountant and
covered with a coverslip. Confocal fluorescence microscopy was
performed at 40.times. magnification with a 1.4 NA oil objective
and 63.times. with a 1.4 NA oil objective. Images were analyzed
with Zen2012 software (Carl Zeiss). Excitation and emission
wavelengths were those recommended by the conjugate manufacturers.
For example, 555 nm was used to excite for phalloidin and
PE-conjugates, 488 nm for Alexa Fluor 488, as well as
FITC-conjugates and 405 nm for DAPI. Emission was detected between
555 and 1000 nm for red fluorescence and 490 nm and above for green
fluorescence. Emission of DAPI was captured below 490 nm or below
529 nm when imaged with red or green fluorescence,
respectively.
[0182] E. Matrigel Invasion Assay, Zymography, and
Immunoblotting
[0183] The ability of 3-BrPA to inhibit tumor invasion was studied
using a matrigel invasion assay, as well as gelatin zymography (Hu
and Beeton (2010) J. Visual. Exp. 45:2445; Scott et al. (2011) J.
Visual. Exp. 58:e3525). For the matrigel invasion assays, a coating
buffer containing 0.01 M Tris and 0.7% sodium chloride was prepared
and used to dilute the matrigel basement membrane (BD Biosciences,
#356234) to 300 ug/ml. Subsequently, Boyden chambers (Transwell,
Corning; 6.5 mm-diameter, 8 um pore size) were coated with 100
.mu.l matrigel solution and stored at 37.degree. C. for 2 hrs. to
allow for gelatination. Approximately 7.5.times.10.sup.4 cells were
resuspended in 500 .mu.l FBS-free medium and plated into the upper
chamber of the insert, which was then placed into a 24-well plate
containing 750 .mu.l of FBS-containing medium. After overnight
incubation at 37.degree. C., various amounts of 3-BrPA dissolved in
PBS were added to the upper chamber. Forty-eight hours later,
non-invasive cells were removed from the matrigel with a cotton
swab. Invaded cells adherent to the bottom side of the permeable
insert were fixed and stained with the Diff Quik Stain Kit
(Polysciences Inc.). Light microscopy was performed using a colored
inverted microscope (Eclipse TS 100) at 4.times., 10.times., and
20.times. magnification. Invasion of cells was quantified by
measuring the area of stained cells after treatment compared to
untreated samples at 10.times. magnification.
[0184] Zymography assays were performed to determine the activity
of secreted MMP-9. Accordingly, 4.times.10.sup.6 Suit-2 cells and
2.5.times.10.sup.6 lucMiaPaCa-2 cells were seeded in 75
cm.sup.2-flasks and incubated overnight at 37.degree. C. under
normoxic conditions. The following day, fresh FBS-free medium
containing different concentrations of 3-BrPA was added and cells
were incubated for an additional 24 hrs. Subsequently, supernatants
were collected, filtered, and the final protein concentrations were
determined using the 2D Quant Kit (GE Healthcare). After adjustment
for concentration, each sample was loaded onto two 10% gelatin
zymography gels (Novex, Invitrogen). Following electrophoresis,
proteins in one of the two gels were renaturated and enzymatic
digestion was allowed overnight at 37.degree. C. in a developing
buffer. The gel was stained with 4 parts 0.1% Coomassie Brilliant
Blue in 1 part 100% methanol for 24 hrs. and washed with distilled
(DI) water until digested areas were detectable as white bands.
Western Blot analysis was performed using the duplicate gel.
Proteins were blotted onto a PVDF-Membrane and blocked using 5%
skimmed milk in 1.times.TBS and 0.1% Tween in DI (TBST). Primary
anti-MMP antibody (Cell Signaling) was used in a 1:1000 dilution
and incubated at 4.degree. C. overnight, followed by an
HRP-conjugated secondary antibody (Santa Cruz) incubation for 1 hr.
at room temperature. The HRP provided an electrochemiluminescence
signal (ECL Kit, GE Healthcare), which was analyzed with ImageJ
1.46r software (Wayne Rasband, National Institute of Health) and
used to quantify signal intensity by comparing line integrals.
[0185] F. Orthotopic Animal Xenografts
[0186] Male athymic nude mice (body weight, 20-25 g; 4 weeks old;
Crl:NU (NCr)-Foxn1.sup.nu; Charles River Laboratory, Germantown,
Md., USA) were used in accordance with institutional guidelines
under approved Animal Care and Use Committee protocols. Mice were
maintained in laminar flow rooms at constant temperature and
humidity with food and water given ad libitum. Orthotopic xenograft
tumors were generated by implantation of 1.5.times.10.sup.6
lucMiaPaCa-2, suspended in 50 .mu.l PBS, into the tail of the
pancreas in anesthetized mice. To accomplish this, mice were placed
into a clean anesthesia induction chamber (oxygen flow rate, 1
liter/minute; isoflurane concentration of 3-4%). Upon loss of the
righting reflex, animals were placed on the surgical procedure
surface, where a nose cone was used to maintain anesthesia (oxygen
flow, 0.8 liters/minute; Isoflurane concentration, 1.5-2%). A
small, left abdominal flank incision was made, and the pancreas was
exteriorized to inject the cell solution using a 30G Hamilton
syringe. A successful subcapsular intrapancreatic injection was
identified by the appearance of a fluid bleb without
intraperitoneal leakage. The abdominal cavity was closed with a
double-layer of non-absorbable suture material (Kim et al. (2009)
Nat. Protocol. 4:1670-1680).
[0187] G. Bioluminescence Imaging and Ultrasound Imaging
[0188] Tumor viability was confirmed via in vivo bioluminescence
imaging (BLI) on day 7 after the surgical implantation.
Anesthetized tumor-bearing mice were injected intraperitoneally
with D-luciferin 150 mg/kg and optically imaged 5 minutes later
using the IVIS 200 system (Xenogen). The pseudocolor image
representing the spatial distribution of photons was overlaid on a
previously acquired grayscale photographic image. A region of
interest (ROI) was created to include the optical tumor image.
Signal intensity was quantified within the ROI in
photons/second/squared centimeter/steradian (p/s/cm2/Sr) after a
10-second exposure using Living Image software (Xenogen).
Additionally, orthotopic growth of the tumors was confirmed prior
to treatment using small-animal ultrasound imaging (USI). In brief,
anesthetized mice were subjected to examination using the VEVO2100
(Visual Sonics Inc., Toronto, Canada, kindly provided by Dr. Harry
C. Dietz) by applying a MS-550D MicroScan transducer probe with 40
MHz (broadband with 22-55 MHz). Tumor localization was confirmed
using the cranial tip of the left kidney and the caudal tip of the
spleen as anatomic landmarks (Ota et al. (2013) Target. Oncol.
8:145-151; Tuli et al. (2012) Translat. Oncol. 5:77-84).
[0189] H. Treatment Regimen and Imaging Follow-Up
[0190] Animals with tumors, as confirmed by both LI and USI, were
randomized into four groups: group 1 (N=21 animals) received daily
intraperitoneal injections of the .beta.-CD-3-BrPA complex (in a
1:1 molecular ratio, 5 mg/kg 3-BrPA in 26 mg/kg .beta.-CD,
dissolved in 500 .mu.l saline), group 2 (N=7 animals) received
intraperitoneal injections of gemcitabine (150 mg/kg dissolved in
200 .mu.l saline, three injections/week as commonly reported in
literature, such as Liau and Whang (2008) Clin. Canc. Res.
14:1470-1477; Larbouret et al. (2010) Annal. Oncol. 21:98-103),
group 3 (N=7) received daily intraperitoneal injections of
.beta.-CD (26 mg/kg .beta.-CD, dissolved in 500 .mu.l saline), and
group 4 (N=7 animals) received daily intraperitoneal injections of
3-BrPA alone (5 mg/kg dissolved in 500 .mu.l saline). All animals
were treated without interruptions for a period of four weeks. BLI
was acquired on day 7, 14, 21, 28 after the first injection.
Animals were sacrificed on day 28 after the last imaging session or
when moribund.
[0191] I. Immunohistochemistry
[0192] Upon sacrificing the animals, tumors were obtained, fixed
with a 4% formaldehyde solution for a period of at least 72 hrs.,
and embedded in paraffin for immunohistochemical analysis.
Hematoxylin and eosin (H&E) staining of the slides was
performed according to standard protocols, such as those described
in Casadonte and Caprioli (2011) Nat. Protocol. 6:1695-1709.
Eighteen .mu.m thick tumor sections were stained for the following
targets: GAPDH, MCT-1, cleaved caspase-3, and Ki-67 using the
Histostain Plus 3rd Gen IHC Detection Kit (Invitrogen), as well as
the Ki-67 kit (Dako Inc.). Specifically, specimens were
deparaffinized using xylene and rehydrated using a descending
ethanol dilution series. After washing with deionized water,
samples were permeabilized in boiling retrieval solution containing
citrate (Dako) for 40 mins. at 95.degree.. Specimens were cooled
down to RT and incubated with 2 drops (.about.100 ul total) of
peroxidase quenching solution for 5 min. and blocked for 20 mins.
Incubation with primary antibodies (GAPDH, 1:500; MCT-1, 1:250;
Ki-67 and HIF-1.alpha.; 1:50, cleaved caspase-3, 1:1,500; in PBS)
occurred at RT in a moist chamber for 60 mins. Biotinylated
secondary antibody and streptavidin-peroxidase conjugate were added
to the samples in sequence for 10 min. each. 26.5 ul of
3,3'-diaminobenzidine (DAB) chromogen were mixed well with 1 ml of
DAB substrate buffer and 100 ul were added to each specimen for 5
mins. Hematoxylin was used as a counterstain. Incubation steps were
followed by washing with distilled water and twice PBS for 2 mins.
each. Samples were sealed using antifading mountant and covered
with a coverslip. All slides were scanned and digitalized at a
20.times. magnification using a high-resolution Aperio.RTM. scanner
system (Vista, Calif., USA). The digitalized slides were then
assessed using the Aperio ImageScope.RTM. software. For the
Ki-67-stained tissue sections, a total of 5-10 fields were viewed
at 10.times., and the number of Ki-67-positive cells, as well as
the total number of cells were recorded to calculate the Ki-67
labeling index (formula: Index [%]=[number of positive cells/total
cell number].times.100).
[0193] J. Statistical Data Analysis
[0194] All experiments were performed independently and repeated at
least three times. Data from the experiments were summarized with
means.+-.standard error of the mean. Statistical comparisons of
data sets were carried out by the Student's t-test as well as the
one-way ANOVA test. Reported BLI signal intensities were normalized
among the animals and reported as multiples based on the baseline
value.
Example 5: .beta.-CD-3-BrPA Shows Strong Anti-Cancer Effects in 2D
and 3D Cell Culture and Targeting Metabolism Reduces Invasive
Potential of Cancer Cells
[0195] Upon NMR-spectroscopic confirmation of the complexation
between 3-BrPA and .beta.-CD (FIG. 7), the microencapsulated
formulation of the drug was used for further experiments. In order
to assess the efficacy of the microencapsulated 3-BrPA
(.beta.-CD-3-BrPA) to achieve dose-dependent ATP depletion and cell
death, two human pancreatic cancer cell lines were employed.
MiaPaCa-2 was derived from a primary pancreatic adenocarcinoma
(PDAC) and Suit-2 was derived from a metastatic primary pancreatic
adenocarcinoma from a different patient (Kitamura et al. (2000)
Clin. Exp. Metast. 18:561-571). The dose-dependent effects of
.beta.-CD-3-BrPA were compared with free 3-BrPA, as well as
gemcitabine, and .beta.-CD was used as a control. As hypoxia is
often associated with chemooresistance in PDACs, hypoxic exposure
was added to the experimental design (Yokoi and Fidler (2004) Clin.
Canc. Res. 10:2299-2306; Kasuya et al. (2011) Oncol. Rep.
26:1399-1406; Onozuka et al. (2011) Canc. Sci. 102:975-982; Zhao et
al. (2014) Canc. Res. 74:2455-2464)). It was found that
.beta.-CD-3-BrPA and free 3-BrPA demonstrated similar cytotoxicity
profiles under normoxic (50-75 .mu.M), as well as hypoxic (12.5-25
.mu.M), conditions and, interestingly, were more sensitive to the
drugs when hypoxic (FIG. 8). Cell lines treated with .beta.-CD
alone were perfectly viable throughout the experiment, even when
exposed to very high concentrations. Similar results were observed
for Suit-2 cells but with less pronounced differences between
normoxic and hypoxic conditions (FIG. 8). When assessing the
efficacy of gemcitabine, IC.sub.50 in MiaPaCa-2 and Suit-2 cells
was barely achieved under normoxic conditions (0.1 .mu.M), no
concentration achieved a complete kill, and hypoxia seemed to
increase the resistance towards the drug.
[0196] In order to test the efficacy of .beta.-CD-3-BrPA in an
ECM-rich environment, lucMiaPaCa-2 cells were cultured in a 3D
Collagen 1 matrix and treated with a single dose of either
.beta.-CD-3-BrPA, free 3-BrPA or .beta.-CD (as a control). BLI
quantification showed that both drug formulations had equivalent
potencies in normoxic conditions (IC.sub.50, 25-50 .mu.M) (FIG. 8).
Under hypoxic conditions, MiaPaCa-2 cells were slightly more
sensitive to free 3-BrPA than to .beta.-CD-3-BrPA (FIG. 8). The
cells cultured in 3D were treated sequentially with the drugs, as
described in Example 4. Morphological, BLI, and
immunofluorescence-based analysis confirmed the ability of 3-BrPA
to penetrate an ECM-rich matrix and to inhibit cell proliferation,
as well as to induce apoptosis (FIGS. 9-10). As such, untreated
MiaPaCa-2 cells proliferated and formed "grape"-like structures
within the collagen 1 matrix, while Suit-2 cells demonstrated a
more invasive growth pattern with cellular protrusions visible
after 6 days of growth (FIG. 10). When treated with 3-BrPA,
proliferation in both cell lines was inhibited with a marked
reduction of cell protrusions in Suit-2 cells (FIG. 10). In
addition, immunofluorescence imaging confirmed a dose-dependent
induction of apoptosis by 3-BrPA.
[0197] In addition, the ability of 3-BrPA to inhibit the
invasiveness of pancreatic cancer cells in sub-lethal drug
concentrations was tested using a matrigel invasion assay. As shown
in FIGS. 11A-11B, both the locally invasive MiaPaCa-2 cells and the
metastatic Suit-2 cells showed a reduction in invasion at drug
concentrations as low as 12.5 .mu.M. In addition, the effect of
sub-lethal doses of 3-BrPA on the secretion of the
matrix-metalloproteinase 9 (MMP-9), a well-described marker for the
invasive potential of pancreatic cancer cells, was tested using
gelatin zymography and immunoblotting (Jones et al. (1999) Annal.
N. Y. Acad. Sci. 880:288-307; Merdad et al. (2014) Anticanc. Res.
34:1355-1366; Yang et al. (2001) J. Surg. Res. 98:33-39).
Accordingly, a marked reduction in the secretion of MMP-9 was
detected in both cell lines. This effect was observed beginning
with a 3-BrPA concentration of 6.25 .mu.M, which is a dose that did
not induce apoptosis or reduce cell viability, and an earlier onset
in the more metastatic Suit-2 cell line (FIGS. 11C-11D).
Example 6: Systemic Delivery of .beta.-CD-3-BrPA Achieves Strong
Anti-Cancer Effects In Vivo
[0198] The anti-cancer efficacy of systemically delivered
.beta.-CD-3-BrPA was tested using a xenograft model of human
pancreatic cancer in athymic nude mice. Prior to choosing the
therapeutic dose for more detailed studies, comparative dose
escalation studies in non-tumor-bearing animals were performed for
both .beta.-CD-3-BrPA and free 3-BrPA. Accordingly, 20 mg/kg of
.beta.-CD-3-BrPA and 10 mg/kg free 3-BrPA were identified as the
median lethal doses (LD.sub.50) after a single injection and 5
mg/kg .beta.-CD-3-BrPA was identified as a safe dose that did not
cause any toxicity when given systemically and daily over the
course of 7 days. A total of 42 animals with
orthotopically-implanted and BLI- and USI-confirmed MiaPaCa-2
tumors were then randomized to receive intraperitoneal (i.p.)
injections of .beta.-CD-3-BrPA (N=21), gemcitabine (N=7), or
.beta.-CD (N=7). An additional group of animals with orthotopic
implants (N=7) was treated with free 3-BrPA. Animals treated with
daily intraperitoneal (i.p.) injections of free 3-BrPA (5 mg/kg in
500 .mu.l saline) demonstrated high treatment-related toxicity and
3/7 (43%) animals died before the acquisition of the first
follow-up BLI (FIG. 12C). At the end of the experiment (Day 28),
only 2/7 animals (28%) treated with the free drug were still alive
(FIG. 12C). No such mortality rate was observed for any of the
remaining groups. Daily i.p. injections of .beta.-CD-3-BrPA (5
mg/kg in 500 .mu.l saline) demonstrated strong anti-cancer effects
with early effects visible on day 14 after the first injection
(FIG. 12B). After four weeks of treatment, a comparison of BLI
signal intensity between the groups was performed. Animals treated
with the .beta.-CD control demonstrated a 140-fold signal increase
as compared to baseline. A moderate deceleration of tumor growth
was observed in gemcitabine-treated animals with a 60-fold signal
increase over time. Most importantly, animals treated with
.beta.-CD-3-BrPA showed minimal or no progression of the signal as
compared to gemcitabine and control groups (FIG. 12). After
achieving this endpoint, animals were sacrificed and tumors were
harvested for further analysis. All animals were subjected to
necropsies and organs (brain, heart, lungs, bowel, liver, and
kidneys) were harvested for the analysis of potential tissue
damage. No organ toxicities or tissue damage was observed in
animals treated with .beta.-CD-3-BrPA (FIG. 12D). The analysis of
tumor pathology demonstrated vast tumor destruction with central
areas of colliquative necrosis in animals treated with
.beta.-CD-3-BrPA (FIG. 13). Tumor regions with intact cell
junctions demonstrated a high expression of cleaved caspase-3,
indicating fulminant tumor apoptosis. Animals treated with
.beta.-CD-3-BrPA demonstrated a significant reduction in
proliferation as assessed with Ki67 immunohistochemistry with a
mean of 17% and 51%, respectively (FIG. 13). In addition, animals
treated with .beta.-CD-3-BrPA demonstrated lower expression levels
of MCT1 and GAPDH within the treated tumors as compared to the
.beta.-CD or gemcitabine groups.
[0199] These results indicate that systemically delivered
.beta.-CD-3-BrPA achieved strong anti-tumoral effects in vivo while
causing much less toxicity in therapeutic doses when compared to
the free drug. Furthermore, microencapsulation of 3-BrPA did not
alter the efficacy of the drug against pancreatic cancer cells in
vitro, which was demonstrated using 2D, as well as ECM-rich 3D cell
cultures, both under normoxic and hypoxic conditions. The abilities
of 3-BrPA to inhibit the secretion of MMP-9 and to reduce the
invasiveness of pancreatic cancer cells in sublethal doses further
indicates the anti-metastatic potential of this drug.
[0200] Selectively targeting tumor metabolism has long been
considered as a desirable therapeutic option, but has yet not been
translated into clinical practice. The primary limitation in
reaching the milestone of systemic deliverability with 3-BrPA is
the reported toxicity due to its alkylating properties
(Ganapathy-Kanniappan and Geschwind (2013) Mol. Cancer 12:152;
Chang et al. (2007) Acad. Radiol. 14:85-92; Kunjithapatham et al.
(2013) BMC Res. Not. 6:277). As a result, local image-guided
delivery of the drug has been explored as an alternative
therapeutic option; however, the practical use of these approaches
is limited to treating localized disease (Ota et al. (2013) Target.
Oncol. 8:145-151; Geschwind et al. (2002) Canc. Res. 62:3909-3913).
The results described herein clearly demonstrate that the drug,
when appropriately formulated for systemic delivery, was extremely
effective, thereby expanding the use of this compound to virtually
any cancer. These results contrast to those of the only other study
where the drug was used systemically in its free form to treat
solid tumors. In that study, free 3-BrPA failed to elicit any
meaningful tumor response at the dose used in the experiments
described herein (Cao et al. (2008) Clin. Canc. Res. 14:1831-1839;
Schaefer et al. (2012) Translat. Res. 159:51-57). Specifically, a
study, which explored the systemic delivery of free 3-BrPA in
combination with an HSP90 inhibitor in subcutaneous pancreatic
cancer xenografts, did not report any significant efficacy for
3-BrPA alone in a dose of 5 mg/kg, given twice per week out of
safety considerations (Cao et al. (2008) Clin. Canc. Res.
14:1831-1839). A possible explanation for this unfavorable efficacy
profile of the free drug is the rapid inactivation of 3-BrPA
through unspecific interaction with serum proteins, which is known
to occur in vivo as early as 2-3 minutes after systemic
administration (Kunjithapatham et al. (2013) BMC Res. Not. 6:277).
Some efficacy was observed at these doses, but excessive toxicity,
with treatment-related deaths in most animals, was the predominant
result. Hence, it is believed that systemic administration of free
3-BrPA may not be effective and may promote undesirable toxicities.
It is also believed that in the microencapsulated formulation,
3-BrPA is more bioavailable for uptake into tumor cells and less
available to the normal cells that apparently mediate its toxicity
(Birsoy et al. (2013) Nature Genet. 45:104-108; Zhang and Ma (2013)
Advanc. Drug Deliv. Rev. 65:1215-1233; Heidel and Schluep (2012) J.
Drug Deliv. 2012:262731).
[0201] A characteristic feature of pancreatic tumor tissue is the
excessive accumulation of dense ECM which limits oxygen diffusion
and creates a highly hypoxic, ill-perfused tumor microenvironment
known for its profound chemoresistance and increased invasiveness
(Yokoi and Fidler (2004) Clin. Canc. Res. 10:2299-2306; Yang et al.
(2001) J. Surg. Res. 98:33-39). Published studies confirmed that
more than 30% of pancreatic tumor cells are located in hypoxic
tumor compartments, thereby escaping the effects of conventional
chemotherapy. These cells then go on to re-form a tumor that has
become even more aggressive and resistant to chemotherapy
(Guillaumond et al. (2013) Proc. Natl. Acad. Sci. U.S.A.
110:3919-3924). The results described herein demonstrate that
3-BrPA is able to effectively block tumor glycolysis even when it
is exacerbated under hypoxic conditions. On the contrary, the
inability of gemcitabine, even at the highest dose, to cope with
hypoxia in pancreatic cancer cells was confirmed. So far,
conflicting data have been reported for the oxygen dependency of
3-BrPA in cancer cells (Cao et al. (2008) Canc. Chemother.
Pharmacol. 62:985-994; Xiao et al. (2013) Oncol. Rep. 29:329-334).
However, there is significant evidence in support of the ability of
3-BrPA to overcome hypoxia as a key mechanism of drug resistance
(Xu et al. (2005) Canc. Res. 65:613-621). Specifically, more recent
studies established the link between hypoxia and the expression of
MCT-1, which was shown to be overexpressed in hypoxic cells and
tumor regions, thus providing the functional explanation for as the
increased sensitivity of hypoxic tumor tissue towards 3-BrPA
(Matsumoto et al. (2013) Magnet. Res. Med. 69:1443-1450). Of note,
combining gemcitabine and 3-BrPA in order to potentially achieve an
increase of efficacy has been explored in vitro; however, no
combination effects were identified and, accordingly, no respective
in vivo experiments were performed.
[0202] Furthermore, the use of the collagen 1-rich 3D organotypic
cell culture as a model for an ECM-rich tumor microenvironment has
demonstrated the ability of 3-BrPA to successfully penetrate the
matrix without any measurable reduction of efficacy as compared to
the monolayer cell culture. The 3D cell culture model used in the
studies described herein can be seen as relatively specific
primarily because it is composed of a matrix, which mimics the
collagen 1-rich ECM as seen in in human ex vivo samples
(Mollenhauer et al. (1987) Pancreas 2:14-24). While the benefits of
such in vitro models for the purpose of drug testing are
increasingly recognized, mimicking these conditions in vivo
represents a greater challenge (Longati et al. (2013) BMC Canc.
13:95). When designing this study, different animal models were
considered. On the one hand, using a widely recognized orthotopic
xenograft model brings about important advantages, such as
reproducibility, predictable tumor growth dynamics, as well as
allowing for genomic modification of tumor cells to express
specific and imageable reporter genes (Kim et al. (2009) Nat.
Protocol. 4:1670-1680). On the other hand, the degree to which
these models reflect the tumor microenvironment in human lesions
remains unknown. Although several well defined mouse tumor models
are able to mimic the ECM-component and tumor hypoxia more
realistically, these models seem as less suitable for the purpose
of standardized drug testing (Guillaumond et al. (2013) Proc. Natl.
Acad. Sci. U.S.A. 110:3919-3924). In light of the demonstrated
ability of 3-BrPA to inhibit cell invasiveness in vitro, the use of
a metastatic Suit-2 xenograft model was considered. However,
orthotopic implantation of Suit-2 xenografts resulted in
complications (i.e., bloody ascites) and loss of a majority of
animals within 14 days after implantation, which facilitated the
selection of MiaPaCa-2 xenografts as a practicable alternative.
[0203] An additional unexpected result was observed in the
immunohistochemical analysis of treated tumor tissues: next to the
anticipated and previously reported depletion of GAPDH as the
molecular target of 3-BrPA, the amount of MCT-1 as the specific
transporter for 3-BrPA was significantly reduced in treated samples
(Ganapathy-Kanniappan et al. (2012) Radiol. 262:834-845). No
evidence has heretofore existed for the presence of MCT-1 as a
potential target of 3-BrPA. Yet, this lactate transporter has been
repeatedly identified as a suitable molecular target of cancer
therapy (Schneiderhan et al. (2009) Gut 58:1391-1398; Shih et al.
(2012) Oncotarget 3:1401-1415; Sonveaux et al. (2012) PloS One
7:e33418).
[0204] Thus, the results described herein identified
microencapsulation of 3-BrPA as a promising advance towards finally
achieving the goal of systemically deliverable anti-glycolytic
tumor therapy. The strong anti-cancer effects of .beta.-CD-3-BrPA
and the favorable toxicity profile pave the way towards clinical
trials in patients with pancreatic cancer and potentially other
malignancies.
INCORPORATION BY REFERENCE
[0205] The contents of all references, patent applications,
patents, and published patent applications, as well as the Figures
and the Sequence Listing, cited throughout this application are
hereby incorporated by reference. It will be understood that,
although a number of patent applications, patents, and other
references are referred to herein, such reference does not
constitute an admission that any of these documents forms part of
the common general knowledge in the art.
EQUIVALENTS
[0206] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *