U.S. patent application number 15/015559 was filed with the patent office on 2016-06-02 for treatment for chemotherapy-induced peripheral neuropathy.
The applicant listed for this patent is Hossein A. Ghanbari, Zhi-Gang Jiang. Invention is credited to Hossein A. Ghanbari, Zhi-Gang Jiang.
Application Number | 20160151339 15/015559 |
Document ID | / |
Family ID | 56078470 |
Filed Date | 2016-06-02 |
United States Patent
Application |
20160151339 |
Kind Code |
A1 |
Ghanbari; Hossein A. ; et
al. |
June 2, 2016 |
Treatment for Chemotherapy-Induced Peripheral Neuropathy
Abstract
The present invention provides methods and compositions for
treating chemotherapy induced peripheral neuropathy. One embodiment
of the present invention is directed to a method of treating
chemotherapy induced peripheral neuropathy by administering to a
patient in need at least one thiosemicarbazone compound.
Inventors: |
Ghanbari; Hossein A.;
(Potomac, MD) ; Jiang; Zhi-Gang; (Gaithersburg,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ghanbari; Hossein A.
Jiang; Zhi-Gang |
Potomac
Gaithersburg |
MD
MD |
US
US |
|
|
Family ID: |
56078470 |
Appl. No.: |
15/015559 |
Filed: |
February 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13848262 |
Mar 21, 2013 |
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15015559 |
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Current U.S.
Class: |
424/450 ;
514/352 |
Current CPC
Class: |
A61K 9/1075 20130101;
A61K 9/127 20130101; A61P 25/02 20180101; A61K 9/0019 20130101;
A61K 31/44 20130101; A61K 9/08 20130101 |
International
Class: |
A61K 31/44 20060101
A61K031/44; A61K 9/08 20060101 A61K009/08; A61K 9/107 20060101
A61K009/107; A61K 9/127 20060101 A61K009/127 |
Claims
1. A method for the treatment of chemotherapy induced peripheral
neuropathy comprising of the step of administering to a patient a
composition comprising at least one thiosemicarbazone compound, or
an analogue thereof.
2. The method of claim 1, wherein the at least one
thiosemicarbazone compound comprises
3-aminopyridine-2-carboxaldehyde thiosemicarbazone (PAN-811).
3. The method of claim 2, wherein the step of administering is
intravenous, intraperitoneal, subcutaneous, intramuscular, topical,
transdermal or oral.
4. The method of claim 2, wherein the composition is an injectable
and/or infusable solution.
5. The method of claim 2, wherein the composition is formulated as
a micro emulsion.
6. The method of claim 2, wherein the composition is formulated as
a liposome.
7. A method for the treatment of chemotherapy-induced peripheral
neuropathy comprising administering to a patient a composition
comprising at least one thiosemicarbazone compound (Formula I), or
an analogue thereof: ##STR00003##
8. The method of claim 7, wherein the at least one
thiosemicarbazone compound comprises the compound of Formula II, or
an analogue thereof: ##STR00004##
9. The method of claim 7, wherein the step of administering is
intravenous, intraperitoneal, subcutaneous, intramuscular, topical,
transdermal or oral.
10. The method of claim 7, wherein the composition is an injectable
and/or infusible solution.
11. The method of claim 7, wherein the composition is formulated as
a micro emulsion.
12. The method of claim 7, wherein the composition is formulated as
a liposome.
Description
BACKGROUND OF THE INVENTION
[0001] Chemotherapy-induced peripheral neuropathy (CIPN) is one of
the most common, serious side effects that can lead to dose
reductions or early discontinuation of chemotherapy, reducing the
efficacy of cancer treatments. It can cause debilitating symptoms
and also significantly impacts the patient's quality of life. An
estimated 30 to 40 percent of cancer patients treated with
chemotherapy experience CIPN.
[0002] The peripheral nervous system (PNS) consists of sensory
neurons running from stimulus receptors that inform the central
nervous system (CNS) of the stimuli, and motor neurons running from
the spinal cord to the effectors that take action. In CIPN, an
anticancer drug could impair both sensory and motor functions. The
symptoms usually start in the hands and/or feet and creep up the
arms and legs. Sometimes it feels like a tingling or numbness.
Other times, it's more of a shooting and/or burning pain or
sensitivity to temperature. It can include sharp, stabbing pain.
CIPN can also lead to hearing loss, blurred vision and change in
taste. CIPN can make it difficult to perform normal day-to-day
tasks like buttoning a shirt, sorting coins in a purse, or walking.
In addition, the motor neuron dysfunction manifest as cramps,
difficulty with fine motor activities (e.g. writing or dialing a
phone), gait disturbances, paralysis, spasms, tremors and
weakness.
[0003] CIPN may result from the use of numerous chemotherapeutic
agents, including, but limited to, Ixabepilone (Ixempra Kit),
arsenic trioxide (Trisenox), cytarabine (Cytosar-U, Depocyt,
generics), etoposide, hexamethylmelamine (altretamine [Hexalen]),
Ifosfamide (Ifex, generics), methotrexate (Trexall, generics),
procarbazine (Matulane) and vinblastine. The chemotherapeutic drugs
that most commonly elicit CIPN include platinum compounds
(cisplatin, carboplatin, oxaliplatin), vincristine, taxanes
(docetaxel, paclitaxel), epothilones (ixabepilone), bortezomib
(Velcade), thalidomide (Thalomid) and lenalidomide.
[0004] For treating the pain associated with CIPN, agents that
appear promising include the antidepressants duloxetine and
venlafaxine, which are both serotonin/norepinephrine-reuptake
inhibitors. Another promising agent is a topical compound of the
muscle-relaxant baclofen, the antidepressant amitriptyline, and the
analgesic ketamine Outside of clinical trials, CIPN symptoms are
commonly managed in a manner similar to other types of nerve
pain--that is, with a combination of physical therapy,
complementary therapies such as massage and acupuncture, and
medications that can include steroids, antidepressants,
anti-epileptic drugs, and opioids for severe pain. But these
therapies have not demonstrated true efficacy for CIPN, and
virtually all of the drugs to treat peripheral neuropathy carry
side effects of their own.
[0005] The actual causes of CIPN, on the cellular and tissue level,
is still largely a matter of speculation. Oxidative stress may play
a key role in CIPN. It was found that antioxidant machinery (e.g.
plasma glutathione (GSH) and .alpha.- and .gamma.-tocopherol
concentrations) of cancer patents with chemotherapy decreased and
the GSH redox state became more oxidized. In a rat model of painful
oxaliplatin-induced neuropathy, oxidative stress was found to be an
important component that mediates pain. In the plasma of
oxaliplatin-treated rats, the increases of carbonylated protein and
thiobarbituric acid reactive substances in the sciatic nerve and
the spinal cord indicated the resultant protein oxidation and
lipoperoxidation in these locations, respectively. Oxidative
imbalance manifests itself as a mediator of inflammatory pain as
well. Use of the anticancer drug cisplatin results in severe cell
death of sensory neurons derived from dorsal root ganglia following
increase in oxidative stress. Oxidative stress is also found to
impair the autonomic nervous system and manifests itself in
symptoms such as hearing loss. The results from antioxidants also
support a key role of oxidative stress in mediating CIPN. The
antineuropathic effect of antioxidant silibinin or
.alpha.-tocopherol shows as about 50% oxaliplatin-induced
behavioral alterations. Administration of anticancer drug
bortezomib or oxaliplatin, which elicits TRPA1-dependent
hypersensitivity, produced a rapid, transient increase in plasma of
carboxy-methyllysine, a by-product of oxidative stress. Short-term
systemic treatment with either HC-030031 or .alpha.-lipoic acid (an
antioxidant) could completely prevent hypersensitivity if
administered before the cytotoxic drug. The findings highlight a
key role for early activation/sensitization of TRPA1 by oxidative
stress by-products in producing CIPN. For preventing the onset of
CIPN, further clinical testing of many antioxidative stress agents,
such as glutathione, acetyl-L-carnitine and alpha-lipoic acid has
been suggested.
[0006] Another mechanism underlying CIPN is excitotoxicity where
increased release of glutamate forces N-methyl D-aspartate (NMDA)
receptors to remain open, allowing increased calcium flux into
neurons, resulting in overexcitation and eventually neuronal
rupture. The end result of this process is pain without a painful
stimulus, also known as neuropathic pain.
N-Acetyl-aspartyl-glutamate (NAAG) is an abundant neuropeptide
widely distributed in the central and peripheral nervous system
which is physiologically hydrolyzed by the enzyme glutamate
carboxypeptidase into N-Acetyl-aspartyl (NAA) and glutamate.
Glutamate carboxypeptidase inhibition could reduce the severity of
chemotherapy-induced peripheral neurotoxicity in rat.
[0007] As there are no proven treatments, there is a need for
methods to properly treat chemotherapy-induced peripheral
neuropathy. The present invention provides just such a method.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to a method of treating
chemotherapy-induced peripheral neuropathy.
[0009] One embodiment of the present invention is directed to a
method of treating chemotherapy-induced peripheral neuropathy by
administering to a patient in need at least one thiosemicarbazone
compound.
[0010] Another embodiment of the present invention is directed to a
method of treating chemotherapy-induced peripheral neuropathy by
administering to a patient in need a composition comprising
3-aminopyridine-2-carboxaldehyde thiosemicarbazone, or an analogue
thereof.
[0011] Another embodiment of the present invention is directed to a
method of treating chemotherapy-induced peripheral neuropathy by
administering to a patient in need a composition comprising
3-aminopyridine-2-carboxaldehyde thiosemicarbazone the step of
administering is intravenous, intraperitoneal, subcutaneous,
intramuscular, topical, transdermal or oral.
[0012] The present invention further encompasses methods of
treating chemotherapy-induced peripheral neuropathy by
administering a composition comprising a compound of Formula I, or
an analogue thereof:
##STR00001##
[0013] Wherein R, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently selected from the group consisting of hydrogen,
C1-8alkyl, C2-8alkenyl, C2-8alkynyl, C3-8cycloalkyl, C1-8haloalkyl,
C6-10aryl, amino-C1-8alkyl, hydroxy-C1-8alkyl,
C1-8alkoxye-C1-8alkyl, and C1-8alkanoyl, or NR.sub.1R.sub.2 taken
in combination form a 3 to 7 member ring which may comprise 0, 1,
or 2 additional ring heteroatoms selected from N, O, and S; R.sub.6
is hydrogen, hydroxy, amino, or C1-8alkyl; R.sub.5 and R.sub.7 are
independently selected from the group consisting of hydrogen,
halide, hydroxy, thiol, amino, hydroxyamino, mono-C1-8alkylamino,
di(C1-8alkyl)amino, C1-8alkoxy, C1-8alkyl, C1-8alkenyl, and
C2-8alkynyl.
[0014] The present invention further encompasses methods of
treating chemotherapy-induced neuropathy by administering a
composition comprising a compound of Formula II, or an analogue
thereof:
##STR00002##
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1: Neurotoxicity of MTX, 5-FU, or CDDP in an
AO-dependent manner. (a, b) Phase contrast photographs for neurons
in 25% AO and 12.5% AO, respectively (bar=25 .mu.m). (c) Cell
membrane leakage was determined via the LDH analysis at the end of
experiment for neurons in 25% AO (n=5). (d) Cell viability was
determined with MTS analysis for neurons in 12.5% AO (n=5). The bar
in green and bars in other colors indicate the cultures without an
insult and with anticancer drug insults, respectively. LDH and MTS
data are expressed as % of noninsulted control. Figure symbol is
##, P<0.01, compared with noninsult control group by Student's
t-test.
[0016] FIG. 2: Dose-dependent neuroprotection of
PAN-811.Cl.H.sub.2O against anticancer drug-induced neurotoxicity.
(a) Phase contrast photographs for neurons in 12.5% AO (bar=25
.mu.m; PAN: PAN-811.Cl.H.sub.2O). (b) LDH analysis for (a) (n=5).
(c) MTS analysis for (a) (n=6). The bar in green and bars in other
colors in the graphs indicate the cultures without an insult and
with anticancer drug insults, respectively. Data are expressed as %
of noninsulted control. Figure symbols are *, P<0.05, and **,
P<0.01, compared with insult group alone (without PAN-811
treatment) by one-factor ANOVA followed with Tukey HSD test.
[0017] FIG. 3: Suppression of 5-FU/MTX-induced increases in LDH and
DHR123 readings by PAN-811. (a) LDH release analysis for neurons
that were cultured in 17.5% AOs containing medium and insulted with
both 100 .mu.MMTX and 25 .mu.M 5-FU in the absence or presence of
PAN-811.Cl.H.sub.2O at different concentrations for 1 day (n=6).
(b) Fluorescent microscope for neurons in 17.5% AOs-containing
medium insulted with both 100 .mu.MMTX and 25 .mu.M 5-FU in the
absence or presence of 10 .mu.M PAN-811.Cl.H.sub.2O for 1 day and
incubated with DHR123 for 30 min (bar=50 .mu.m). (c) Quantification
of (b) at excitation at 485 nm and emission at 520 nm (n=4). Data
are expressed as %
suppression=[(Insulted&Untreated-Insulted&Treated)/(Insulted&Untreated-No-
nInsulted&Untreated)*100%]. Figure symbols are *, P<0.05,
compared with insult group alone (without PAN-811 treatment) by
Student's t-test (one tail) and one-factor ANOVA followed by Tukey
HSD test; #, P<0.05; ##, P<0.01, compared with
noninsult/untreated control group by one-factor ANOVA followed with
Tukey HSD test.
[0018] FIG. 4: No interference of PAN-811.Cl.H.sub.2O with
anticancer drug-induced cytotoxicity to mouse cancer cell line
BNLT3. (a)-(c) Phase contrast photographs for BNLT3 cells that were
treated without or with 10 .mu.MPAN-811.Cl.H.sub.2O and insulted
with 100 .mu.MMTX, 25 .mu.M5-FU, and 3.5 .mu.M CDDP for 3 days,
respectively (bar=50 .mu.m). (d)-(f) MTX analysis corresponding to
(a)-(c) (n=6). Data are expressed as % of noninsulted/untreated
control. Figure symbol is ##, P<0.01, compared with
noninsult/untreated control group by one-factor ANOVA followed with
Tukey HSD test.
[0019] FIG. 5: Effects of PAN-811.Cl.H.sub.2O on anticancer
drug-induced cytotoxicities to human cancer cell H460. (a) Phase
contrast photographs for the H460 cells that received 25 .mu.M
5-FU, 10 .mu.M PAN-811, or both for 3 days (bar=50 .mu.m). (b)-(d)
MTS analysis for the H460 cells that received 10 .mu.M
PAN-811.Cl.H2O, one of 100 .mu.M MTX, 25 .mu.M 5-FU, and 3.5 .mu.M
CDDP, or both 10 .mu.M PAN-811.Cl.H.sub.2O and one of these
anticancer drugs for 3 days, respectively (n=6). (e)-(g) LDH
analysis for (b)-(d) (n=6). Data are expressed as % of
noninsulted/untreated control. Figure symbol is ##, P<0.01,
compared with noninsult/untreated control group by one-factor ANOVA
followed with Tukey HSD test.
DETAILED DESCRIPTION OF THE INVENTION
[0020] For simplicity and illustrative purposes, the principles of
the present invention are described by referring to various
exemplary embodiments thereof. Although the preferred embodiments
of the invention are particularly disclosed herein, one of ordinary
skill in the art will readily recognize that the same principles
are equally applicable to, and can be implemented in other systems,
and that any such variation would be within such modifications that
do not part from the scope of the present invention. Before
explaining the disclosed embodiments of the present invention in
detail, it is to be understood that the invention is not limited in
its application to the details of any particular arrangement shown,
since the invention is capable of other embodiments. The
terminology used herein is for the purpose of description and not
of limitation. Further, although certain methods are described with
reference to certain steps that are presented herein in certain
order, in many instances, these steps may be performed in any order
as would be appreciated by one skilled in the art, and the methods
are not limited to the particular arrangement of steps disclosed
herein.
[0021] The present invention is directed to a method for the
treatment of chemotherapy induced peripheral neuropathy comprising
the step of administering to a patient a composition comprising a
thiosemicarbazone compound. The means for synthesis of
thiosemicarbazone compounds useful in the methods of the invention
are well known in the art. Such synthetic schemes are described in
U.S. Pat. Nos. 5,281,715; 5,767,134; 4,447,427; 5,869,676 and
5,721,259; all of which are incorporated herein by reference in
their entirety.
[0022] The chemical structures of PAN-811's analogues are shown in
U.S. Pat. No. 7,456,179, and patent applications of 20090275587,
20060194810 and 20060160826 each of which are hereby incorporated
by reference.
[0023] The pharmaceutical compositions required by the present
invention typically comprise a compound useful in the methods of
the invention and a pharmaceutically acceptable carrier. As used
herein "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. The type of carrier can be selected
based upon the intended route of administration. In various
embodiments, the carrier is suitable for intravenous,
intraperitoneal, subcutaneous, intramuscular, topical, transdermal
or oral administration. Pharmaceutically acceptable carriers
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the pharmaceutical compositions of
the invention is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0024] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, monostearate salts and gelatin.
Moreover, the compounds can be administered in a time release
formulation, for example in a composition which includes a slow
release polymer. The active compounds can be prepared with carriers
that will protect the compound against rapid release, such as a
controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
polylactic acid and polylactic, polyglycolic copolymers (PLG). Many
methods for the preparation of such formulations are generally
known to those skilled in the art.
[0025] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0026] Depending on the route of administration, the compound may
be coated in a material to protect it from the action of enzymes,
acids and other natural conditions which may inactivate the agent.
For example, the compound can be administered to a subject in an
appropriate carrier or diluent co-administered with enzyme
inhibitors or in an appropriate carrier such as liposomes.
Pharmaceutically acceptable diluents include saline and aqueous
buffer solutions. Enzyme inhibitors include pancreatic trypsin
inhibitor, diisopropylfluoro-phosphate (DEP) and trasylol.
Liposomes include water-in-oil-in-water emulsions as well as
conventional liposomes. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations may contain a preservative to prevent the growth of
microorganisms.
[0027] The active agent in the composition (i.e., one or more
thiosemicarbazones) preferably is formulated in the composition in
a therapeutically effective amount. A "therapeutically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired therapeutic result to
thereby influence the therapeutic course of a particular disease
state. A therapeutically effective amount of an active agent may
vary according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the agent to elicit a
desired response in the individual. Dosage regimens may be adjusted
to provide the optimum therapeutic response. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the agent are outweighed by the therapeutically
beneficial effects. In another embodiment, the active agent is
formulated in the composition in a prophylactically effective
amount. A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically, since a prophylactic
dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically effective amount will be less than
the therapeutically effective amount.
[0028] The amount of active compound in the composition may vary
according to factors such as the disease state, age, sex, and
weight of the individual. Dosage regimens may be adjusted to
provide the optimum therapeutic response. For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0029] A compound of the invention can be formulated into a
pharmaceutical composition wherein the compound is the only active
agent therein. Alternatively, the pharmaceutical composition can
contain additional active agents. For example, two or more
compounds of the invention may be used in combination.
[0030] 3-aminopyridine-2-carboxaldehyde thiosemicarbazone
(hereinafter "PAN-811"), with a molecular weight of 195.24 Da, has
demonstrated potent neuroprotective activities in several
neurodegenerative models. PAN-811 was originally developed for
cancer therapy due to its ability to inhibit ribonucleotide
reductase, a key enzyme required for DNA synthesis. Our previous
studies demonstrated that PAN-811 at concentration of 0.45 .mu.M
fully blocked ischemic neurodegeneration and at 1.2 .mu.M
completely halted hypoxia-induced neuronal cell death. PAN-811 was
administered intracerebroventricularly (i. c. v.) at a dose of 50
.mu.g per rat at 1 h after arterial occlusion. Staining of
consecutive brain sections and computer-assisted quantitative
analysis demonstrated that PAN-811 reduced the infarct volume by
59% in PAN-811 treated rats. We also investigated the effect of a
single intravenous (i. v.) bolus injection of PAN-811. Two-hour
middle cerebral artery occlusion (MCAo) with cerebral blood flow
reduction of 75% or greater resulted in infarct formation, brain
edema and a significant number of premature deaths. PAN-811
treatment reduced infarct volume in a dose dependent manner with a
maximal protection of 50% at a dose of 2 mg/kg. PAN-811 treatment
(2 mg/kg) also resulted in a 70% reduction in brain edema volume.
Accordingly, the mortality in PAN-811 treated groups was
collectively reduced by 44% (Jiang et al., 2008). Mechanistically
PAN-811 prevents glutamate-induced excitatory cytotoxicity,
veratridine-induced sodium channel opening that is related to
Ca.sup.2+ influx and staurosporine-induced apoptosis. Nearly
complete neuroprotection against glutamate insult is observed in
cultured neuronal cells if the cells were pretreated with 10 .mu.M
PAN-811 for 24 h. In culture, ischemic condition results in a
19-fold increase in intracellular free calcium. PAN-811 at a dose
of 5 .mu.M reduced this elevated level by 72%. In a cell-free
system by taking EDTA as a positive control, PAN-811 chelates free
calcium as efficiently as EDTA. In addition, PAN-811 effectively
suppresses oxidative stress in many ways. PAN-811 at a
concentration as low as 1 .mu.M suppressed in vitro hydrogen
peroxide-induced LDH release by 78% (with P<0.01, compared to
untreated/H.sub.2O.sub.2-insulted group) and at a concentration of
10 .mu.M achieved maximal protection (by 90% comparing with
untreated and H.sub.2O.sub.2-insulted group) with an EC.sub.50 of
.about.0.55 .mu.M. PAN-811 also inhibited oxidative stress-induced
cell death of human Alzheimer's disease-derived and age-matched
olfactory neuroepithelial cells via suppression of intracellular
reactive oxygen species. Importantly, PAN-811 manifested as a free
radical scavenger in a cell free system where PAN-811 reduced 500
.mu.M of a stable free radical diphenylpicrylhydrazyl by 70%. Taken
together, PAN-811 has manifested as a potent neuroprotectant with
dual drug targets--oxidative stress and free calcium.
[0031] Based on the key roles of excitoneurotoxicity and oxidative
stress in chemotherapy-induced peripheral neuropathy and also the
potent free calcium chelating and antioxidative effects of PAN-811,
we have discovered that PAN-811 is a therapeutic agent for
chemotherapy-induced peripheral neuropathy. PAN-811 should inhibit
chemotherapy-induced peripheral neuropathy that is not only caused
with antimetabolites (cytarabine, gludarabine, fluorouracil,
mercaptopurine, methotrexate, thioguanine, gemcitabine,
hydroxyurea), mitotic inhibitors (vincristine, vinblastine,
vinorelbine), topoisomerase inhibitors (topotecan, irenotecan),
paclitaxel, docetaxel and asparaginase, but also with alkylating
agents (busulfan. carmustine, lomustine, chlorambucil,
cyclophosphamide, cisplatin, carboplatin, oxaliplatin, ifosamide,
mechlorethamine, melphalan, thiotepa, dacarbazine, procarbazine),
antitumor antibiotics (bleomycin, dactinomycin, daunorubicin,
doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin),
topoisomerase II inhibitor (etoposide, teniposide), and radiation
therapy. In addition, PAN-811 should inhibit chemotherapy-induced
peripheral neuropathy caused by other anticancer drug, such as
ixabepilone, arsenic trioxide, etoposide, hexamethylmelamine,
ifosfamide, methotrexate, procarbazine, epothilones, bortezomib,
thalidomide and lenalidomide.
Example 1
Materials and Methods
[0032] Neuronal Cell Culture. Mixed cortical and striatal neurons
fromembryonic day 17 male Sprague-Dawley rats (tissue obtained from
NIH) were seeded into poly-D-lysine coated 96-well plates at
density of 50,000 cells/well and initially cultured at 37.degree.
C., 5% CO2, in neurobasal medium (NB) with B27 supplement
(Invitrogen) containing full strength of AOs to obtain highly
enriched (95%) neurons. Since AOs, including vitamin E, vitamin E
acetate, superoxide dismutase (SOD), catalase (CAT), and GSH, are
additives to culture medium, reduction of AO concentration in
culture medium provides an approach to determine the level of OS
involvement in a neurotoxic process. In our study, the culture
medium was replaced at a 50% ratio with NB plus B27 minus AOs twice
at days 7 and 9 to set AO concentrations as 50% and 25%,
respectively. At 16 days in vitro (d.i.v.), a fraction of the
culture medium was harvested for lactate dehydrogenase (LDH) assay,
and then AO concentration was reduced to 12.5% and cultured for a
further 5 hours prior to ending the experiment.
[0033] Cancer Cell Culture. The mouse liver cancer cell line BNLT3
(gift of Dr. Jack Wands, Brown University) and the human lung
cancer cell line H460 (ATCC) were seeded into 96-well plates at a
density of 4,000 cells/well and cultured at 37.degree. C., 5% CO2,
in DMEM (11965, Gibco) supplemented with 10% fetal bovine serum, 20
mM HEPES, 1 mM sodium pyruvate, and 24 ng/mL gentamycin (all
reagents came from Gibco).
[0034] Cell Insults and Treatments. Determination of concentration
for each anticancer drug in our experiments was based on its
reported concentration in human cerebral spinal fluid (CSF) in
chemotherapy, literature report of its neurotoxicity in culture,
and our preliminary in vitro experimental data. At 13 d.i.v., the
neuronal cell cultures were insulted with 100 .mu.M of MTX (M9929,
Sigma), 25 .mu.M of 5-FU (F6627, Sigma), or 3.5 .mu.M of CDDP
(sc-200896, Santa Cruz) for 3 days in absence or presence of PAN
811.Cl.H2O. For ROS examination, the neurons were insulted with
both 100 .mu.M of MTX and 25 .mu.M of 5-FU by 15 d.i.v.
PAN-811.Cl.H2O was added to cultures to final concentrations of
1.25, 2.5, 5, and 10 .mu.M at the same time as addition of the
anticancer drugs. For cancer cell lines, the cells were insulted by
the second day of cell seeding with the same concentration of MTX,
5-FU, or CDDP as used in neuronal culture in the absence or
presence of 10 .mu.M PAN-811.Cl.H2O for another 3 days.
[0035] Quantitative Assays and Morphological Assessment. Cell
membrane integrity and mitochondrial function of either neurons or
cancer cells were measured with LDH and
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium [MTS] analyses, respectively. The latter has been
used to quantify cell survival. For the LDH assay, a mixture of a
35 .mu.L aliquot of culture supernatant and 17.5 .mu.L of Mixed
Substrate, Enzyme and Dye Solutions (Sigma) was incubated at room
temperature (RT) for 30 minutes. For the MTS assay, 10 .mu.L of MTS
reagent (Promega) was added to a culture well containing neurons in
50 .mu.L of medium. The preparations were incubated at 37.degree.
C. for 2 hours. The preparations for both assays were then
spectrophotometrically measured at 490 nm using a 96-well plate
reader (Mode 550, Bio-Rad). Neuronal cell death was morphologically
determined based on the integrity of the cell soma and continuity
of neuronal processes. The change in number of cancer cells was
judged directly by cell density. Cells were photographed under an
inverted phase contrast microscope (IX 70, Olympus) using 10.times.
or 20.times. objective.
[0036] ROS Examination. Neurons were incubated in 15 .mu.M
dihydrorhodamine 123 (DHR 123, Molecular Probes) for 30 min at
37.degree. C. to determine intramitochondrial ROS levels.
Fluorescence was photographed by using a fluorescentmicroscope and
quantified by excitation at 485 nm and emission at 520 nm using a
96-well plate reader (Model 550, Bio-Rad).
[0037] Data Analysis. Data were generated from 4-6 replicate wells,
expressed as mean.+-.standard deviation (SD), and statistically
evaluated at a significance level of 1% with onefactor ANOVA or
Student's t-test by using software VASSARSTATS
(http://vassarstats.net/) followed by the Tukey HSD test. Figure
symbols are as follows: #, P<0.05, and ##, P<0.01, compared
with control; *, P<0.05, and **, P<0.01, compared with the
insulted group; .sctn..sctn., P<0.01, compared with PAN-811
treated group by Student's t-test.
Results
[0038] MTX, 5-FU, or CDDP Elicited Neurotoxicity in an
AOs-Dependent Manner. By 3 days following the insults, neither MTX
at 100 .mu.M, 5-FU at 25 .mu.M, nor CDDP at 3.5 .mu.M caused
morphological changes, LDH release, or MTS reduction when neurons
were cultured in the medium containing 100% or 50% AOs (data not
shown). However, MTX, 5-FU, or CDDP at the same concentrations
elicited significantLDHincrease (indicating cellmembrane leakage,
FIG. 1(c)) in the culture supernatant when the AO concentration was
reduced to 25%, although no cell damage was visible (FIG. 1(a)),
and no change in MTS level was detectable (data not shown) under
these conditions. When the AO concentration was reduced to 12.5%
for 5 hours at 16 d.i.v., extensive neuronal cell death occurred in
the cultures insulted with 25 .mu.M 5-FU or 3.5 .mu.M CDDP, as
indicated by loss of cell bodies, together with interruption of
neurite networks on the background (FIG. 1(b)). Corresponding to
the morphological cell death, the MTS readings for 5-FU- and
CDDP-insulted groups were reduced by 27% and 66%, respectively
(FIG. 1(d)).MTX at 100 .mu.M did not elicit significant MTS
reduction (FIG. 1(d)) under 12.5% AO condition. Thus, the
neurotoxicities elicited with MTX, 5-FU, or CDDP were dependent on
AO reduction.
[0039] PAN-811 Dose-Dependently Suppresses MTX-, 5-FU-, or
CDDP-Induced Neurotoxicity. We then examined PAN-811 for its effect
on the anticancer drug-induced neurotoxicity at the 12.5% AO
condition. MTX at 100 .mu.M did not result in significant loss of
cell number, while 5-FU at 25 .mu.M or CDDP at 3.5 .mu.M caused
robust loss of neurons in culture (FIG. 2(a)). Correspondingly, MTX
insult did not significantly affect the MTS reading, while 5-FU-
and CDDPinsulted cultures showed significant reduction in MTS
readings (FIG. 2(c)). PAN-811 dose-dependently inhibited 5-FU- or
CDDP-induced MTS reduction. PAN-811 at 10 .mu.M completely blocked
5-FU-inducedMTS reduction and inhibited CDDP-induced MTS reduction
by 48%. The LDH release assay demonstrated that each of MTX at 100
.mu.M, 5-FU at 25 .mu.M, and CDDP at 3.5 .mu.M resulted in
significant increases in LDH reading (FIG. 2(b)). PAN-811
dose-dependently suppressed LDH increase caused by each anticancer
drug. PAN-811 at 5 .mu.M fully blocked LDH release in MTX-, 5-FU-,
or CDDP-insulted cultures (with no statistically significant
difference from untreated control culture by ANOVA analysis). Thus,
PAN-811 was demonstrated as a potential neuroprotective compound
for anticancer drugMTX-, 5-FU-, or CDDP-induced neurotoxicity.
[0040] PAN-811 Suppresses Cell Membrane Leakage When MTX and 5-FU
Are Coadministered. Since MTX and 5-FU are coadministered for
cancer therapies in many cases, we were interested to know if
PAN-811 can block neurotoxicity that is elicited with a combined
insult with both MTX and 5-FU. An insult with a combined 100 .mu.M
MTX and 25 .mu.M 5-FU resulted in a 109% increase in LDH reading by
comparison with noninsulted control group (P<0.05 by ANOVA; FIG.
3(a)). PAN-811 showed concentration-dependent suppression of LDH
release within the tested range from 1.25 to 10 .mu.M. PAN-811 at
10 .mu.M fully inhibited MTX/5-FU-elicited LDH increase.
[0041] PAN-811 InhibitsMTX- and 5-FU-Elicited OS. To understand the
underlying mechanism for MTX- and 5-Fu induced neurotoxicity, a
cell-permeable fluorogenic probe DHR 123 was used for the detection
of intramitochondrial ROS. Neuronal insult with coadministered 100
.mu.M MTX and 25 .mu.M 5-FU greatly increased intensity of DHR 124
fluorescence (FIG. 3(b)), resulting in a 33.4% increase in DHR123
level in comparison with noninsulted group (P<0.05 by t-test,
data not shown). PAN-811 at 10 .mu.M provided significant
suppression to the increased ROS, showing a 62.3% suppression rate
(FIG. 3(c)).
[0042] PAN-811 Shows No Antagonistic Effect on MTX-, 5-FU-, or
CDDP-Induced Cytotoxicity in BNLT3 Cells. To understand whether
PAN-811 could interfere with anticancer efficacy of tested
anticancer drugs, the mouse liver cancer cell line BNLT3 was
cotreated with each anticancer drug at the concentrations used for
elicitation of neurotoxicity and 10 .mu.M PAN-811, the highest
concentration used for neuronal protection in these experiments. A
3-day insult with 100 .mu.M MTX severely reduced the cancer cell
number (FIG. 4(a)). In the culture treated with 10 .mu.M PAN-811
alone or cotreated with 100 .mu.M MTX and 10 .mu.M PAN-811, cell
density was also much lower than that in no-insult control.
Quantitatively, MTX at 100 .mu.M reduced MTS reading by 85% (FIG.
4(d)), while PAN-811 at 10 .mu.M reduced MTS reading to the same
level as MTX. A cotreatment with both did not cause any further
reduction in MTS reading when comparing with MTX alone. Similarly,
5-FU at 25 .mu.M significantly reduced the cell density of the
cancer cells, and a cotreatment with both 25 .mu.M 5-FU and 10
.mu.M PAN-811 significantly decreased the cell number as well (FIG.
4(b)). Quantitatively, 5-FU at 25 .mu.M reduced MTS reading by 84%,
which was less efficient than 10 .mu.MPAN-811 group (FIG. 4(e)). A
cotreatment with both caused a further reduction in MTS reading
when comparing with 5-FU alone. No synergistic effect between 5-FU
and PAN-811 could be detected. An insult with 3.5 .mu.M CDDP also
caused a decrease in the cell density (FIG. 4(c)), while a
treatment with PAN-811 alone or a cotreatment with both 3.5 .mu.M
CDDP and 10 .mu.M PAN-811 introduced a significant reduction in the
cell density. Quantitatively, 3.5 .mu.M CDDP reduced MTS reading by
44%, while 10 .mu.MPAN-811 caused a 94% reduction in MTS reading
(FIG. 4(f)). A cotreatment with both did not introduce an extra
reduction in MTS reading by comparing with PAN-811 alone, despite
showing much lower reading than CDDP alone (P<0.01). In general,
PAN-811 did not show any inhibition in the effect of MTX, 5-FU, or
CDDP on BNLT3 cells, neither did it demonstrate any synergistic
effect with each tested anticancer drug on BNLT3 cell growth.
[0043] PAN-811 Shows No Antagonistic Effect on MTX-, 5-FU-, or
CDDP-Induced Cell Death of H460 Cells, While Demonstrating a
Synergistic Effect with 5-FU or CDDP on Suppression of the Cell
Growth. To understand whether there is any negative effect of
PAN-811 on the efficacy of tested anticancer drugs in humans, the
human lung cancer cell line H460 was treated with each of these
anticancer drugs at the concentrations used for elicitation of
neurotoxicity, in the absence or presence of 10 .mu.M PAN-811.
A3-day insult with 100 .mu.MMTX, 10 .mu.MPAN-811, or both robustly
decreased the cell density of H460 in culture (data not shown).
Quantitatively, 100 .mu.M MTX and 10 .mu.M PAN-811 reduced MTS
readings by 67% and 76%, respectively. The MTS reading for a
cotreatment with both 100 .mu.MMTX and 10 .mu.M PAN-81 was about
the same as 10 .mu.M PAN-811 alone (FIG. 5(b)). In membrane
integrity analysis (FIG. 5(e)), 100 .mu.M MTX resulted in a 95%
increase in LDH reading in the culture supernatant, while 10 .mu.M
PAN-811 led to a 31% increase in the LDH reading. A cotreatment
with 100 .mu.M MTX and 10 .mu.M PAN-811 reduced LDH reading by 70%
when compared with MTX group (P<0.01 by ANOVA), indicating an
inhibitory effect of PAN-811 on MTX-caused membrane leakage.
Similarly, a 3-day treatment with 25 .mu.M 5-FU, 10 .mu.M PAN-811,
or both robustly decreased the cell density of H460 in culture
(FIG. 5(a)). Quantitatively, 25 .mu.M 5-FU and 10 .mu.M PAN-811
reduced MTS readings by 57% and 74%, respectively (FIG. 5(c)). In
contrast, a cotreatment with 25 .mu.M 5-FU and 10 .mu.M PAN-811
reduced MTS readings by 84%, which shows a statistically
significant difference from 5-FU (P<0.01) or PAN-811 alone
(P<0.01), indicating a synergistic effect of 5-FU and PAN-811 on
suppression of growth of human lung cancer cell H460. In membrane
integrity analysis (FIG. 5(f)), 25 .mu.M5-FU resulted in a 124%
increase in LDH reading in the culture supernatant, while 10 .mu.M
PAN-811 led to a 30% increase in the LDH reading. A cotreatment
with 100 .mu.M5-FU and 10 .mu.MPAN-811 enhanced LDH reading by 40%,
which is much lower than that in the group with 25 M5-FU alone. It
indicates an inhibitory effect of PAN-811 on 5-FU-caused membrane
leakage. A 3-day treatment with 3.5 .mu.M CDDP, 10 .mu.M PAN-811,
or both greatly decreased the cell density of H460 in culture.
Quantitatively, 3.5 .mu.M CDDP and 10 .mu.M PAN-811 reduced MTS
readings by 22% and 75%, respectively (FIG. 5(d)). A cotreatment
with 3.5 .mu.M CDDP and 10 .mu.M PAN-811 reduced MTS readings by
85%, which shows a statistically significant difference from CDDP
(P<0.01 by ANOVA) or PAN-811 alone (P<0.01 by t-test),
indicating a synergistic effect of CDDP and PAN-811 on suppression
of growth of human lung cancer cell H460. In membrane integrity
analysis (FIG. 5(g)), 3.5 .mu.M CDDP resulted in a 71% increase in
LDH reading in the culture supernatant, while 10 .mu.M PAN-811 led
to a 30% increase in the LDH reading. A cotreatment with 3.5 .mu.M
CDDP and 10 .mu.MPAN-811 enhanced LDH reading by 57%, which is a
statistically significant difference from that in the group with
3.5 .mu.M CDDP alone (P<0.01), demonstrating an inhibitory
effect of PAN-811 on CDDP-induced membrane leakage. In general,
PAN-811 did not show any inhibition in the effect of MTX, 5-FU, or
CDDP on cell growth of H460 cells, although it manifested an
inhibitory effect on MTX-, 5-FU-, or CDDP-induced membrane leakage.
A synergistic effect between 5-FU and PAN-811 or between CDDP and
PAN-811 occurred on suppression of H460 cell survival.
[0044] While the invention has been described with reference to
certain exemplary embodiments thereof, those skilled in the art may
make various modifications to the described embodiments of the
invention without departing from the scope of the invention. The
terms and descriptions used herein are set forth by way of
illustration only and not meant as limitations. In particular,
although the present invention has been described by way of
examples, a variety of compositions and processes would practice
the inventive concepts described herein. Although the invention has
been described and disclosed in various terms and certain
embodiments, the scope of the invention is not intended to be, nor
should it be deemed to be, limited thereby and such other
modifications or embodiments as may be suggested by the teachings
herein are particularly reserved, especially as they fall within
the breadth and scope of the claims here appended. Those skilled in
the art will recognize that these and other variations are possible
within the scope of the invention as defined in the following
claims and their equivalents.
* * * * *
References