U.S. patent application number 15/220596 was filed with the patent office on 2016-11-17 for use of psoralen derivatives and combination therapy for treatment of cell proliferation disorders.
This patent application is currently assigned to IMMUNOLIGHT, LLC. The applicant listed for this patent is DUKE UNIVERSITY, IMMUNOLIGHT, LLC. Invention is credited to Wayne F. Beyer, JR., David Gooden, Erik J. Soderblom, Neil L. Spector, Eric J. Toone, Harold Walder, Wenle XIA.
Application Number | 20160331731 15/220596 |
Document ID | / |
Family ID | 53543862 |
Filed Date | 2016-11-17 |
United States Patent
Application |
20160331731 |
Kind Code |
A1 |
XIA; Wenle ; et al. |
November 17, 2016 |
USE OF PSORALEN DERIVATIVES AND COMBINATION THERAPY FOR TREATMENT
OF CELL PROLIFERATION DISORDERS
Abstract
Methods for the treatment of a cell proliferation disease or
disorder in a subject, involving applying a psoralen derivative
lacking a DNA cross-linking motif to cancer cells, applying a
psoralen or a derivative thereof and lapatinib, or applying a
psoralen or derivative thereof and neratinib, to a subject and
further applying initiation radiation energy form an energy
source.
Inventors: |
XIA; Wenle; (Durham, NC)
; Gooden; David; (Durham, NC) ; Soderblom; Erik
J.; (Durham, NC) ; Toone; Eric J.; (Durham,
NC) ; Spector; Neil L.; (Durham, NC) ; Beyer,
JR.; Wayne F.; (Belville, NC) ; Walder; Harold;
(Belville, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IMMUNOLIGHT, LLC
DUKE UNIVERSITY |
Detroit
Durham |
MI
NC |
US
US |
|
|
Assignee: |
IMMUNOLIGHT, LLC
Detroit
MI
DUKE UNIVERSITY
Durham
NC
|
Family ID: |
53543862 |
Appl. No.: |
15/220596 |
Filed: |
July 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14603539 |
Jan 23, 2015 |
9439897 |
|
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15220596 |
|
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61930717 |
Jan 23, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4709 20130101;
A61K 31/436 20130101; A61K 31/517 20130101; A61K 31/4709 20130101;
A61P 35/00 20180101; A61K 31/517 20130101; A61K 31/37 20130101;
A61K 2300/00 20130101; A61K 41/0066 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/436
20130101; A61K 45/06 20130101; A61K 31/37 20130101 |
International
Class: |
A61K 31/436 20060101
A61K031/436; A61K 31/4709 20060101 A61K031/4709; A61K 41/00
20060101 A61K041/00; A61K 45/06 20060101 A61K045/06 |
Claims
1. A pharmaceutical composition for treatment of a cell
proliferation disorder or disease, comprising: a psoralen
derivative lacking a DNA cross-linking motif; and a
pharmaceutically acceptable carrier, wherein the psoralen
derivative lacking a DNA cross-linking motif is represented by the
formula (1): ##STR00006## wherein R.sub.1 is hydrogen, lower alkyl,
or lower alkoxy; R.sub.2 and R.sub.3 are each, independently,
hydrogen, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy, or
R.sub.2 and R.sub.3 may join to form a substituted or
unsubstituted, condensed 5 to 7 membered aliphatic or aromatic
ring, optionally containing at least one heteroatom selected from
N, S, and O; R.sub.4 and R.sub.5 are each, independently, hydrogen,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy, or R.sub.4 and
R.sub.5 may join to form a substituted or unsubstituted, condensed
5 to 7 membered aliphatic or aromatic ring, optionally containing
at least one heteroatom selected from N, S, and O; with the proviso
that at least one of R.sub.2 and R.sub.3 or R.sub.4 and R.sub.5 are
joined to form a substituted or unsubstituted, condensed 5 to 7
membered aliphatic or aromatic ring, optionally containing at least
one heteroatom selected from N, S, and O.
2. The pharmaceutical composition of claim 1, further comprising at
least one substituted psoralen compound selected from the group
consisting of 8-Methoxypsoralen (8-MOP) and
4'-aminomethyl-4,5',8-trimethylpsoralen (AMT).
3. The pharmaceutical composition of claim 1, wherein the psoralen
derivative lacking a DNA cross-linking motif is SMSF032310.
4. The pharmaceutical composition of claim 1, further comprising at
least one energy modulation agent that converts an initiation
energy to an energy that activates the psoralen lacking the DNA
cross-linking motif.
5. The pharmaceutical composition of claim 2, further comprising at
least one energy modulation agent that converts an initiation
energy to an energy that activates one or both of the psoralen
lacking the DNA cross-linking motif or the substituted psoralen
compound.
6. The pharmaceutical composition of claim 3, further comprising at
least one energy modulation agent that converts an initiation
energy to an energy that activates SMSF032310.
7. The pharmaceutical composition of claim 4, wherein the at least
one energy modulation agent is one or more members selected from
the group consisting of biocompatible fluorescing metal
nanoparticles, fluorescing dye molecules, gold nanoparticles, water
soluble quantum dots encapsulated by polyamidoamine dendrimers, a
luciferase, biocompatible phosphorescent molecules, combined
electromagnetic energy harvester molecules, and lanthanide chelates
capable of intense luminescence.
8. The pharmaceutical composition of claim 5, wherein the at least
one energy modulation agent is one or more members selected from
the group consisting of biocompatible fluorescing metal
nanoparticles, fluorescing dye molecules, gold nanoparticles, water
soluble quantum dots encapsulated by polyamidoamine dendrimers, a
luciferase, biocompatible phosphorescent molecules, combined
electromagnetic energy harvester molecules, and lanthanide chelates
capable of intense luminescence.
9. The pharmaceutical composition of claim 6, wherein the at least
one energy modulation agent is one or more members selected from
the group consisting of biocompatible fluorescing metal
nanoparticles, fluorescing dye molecules, gold nanoparticles, water
soluble quantum dots encapsulated by polyamidoamine dendrimers, a
luciferase, biocompatible phosphorescent molecules, combined
electromagnetic energy harvester molecules, and lanthanide chelates
capable of intense luminescence.
10. The pharmaceutical composition of claim 7, wherein the at least
one energy modulation agent is one or more members selected from
the group consisting of biocompatible phosphorescent molecules.
11. The pharmaceutical composition of claim 8, wherein the at least
one energy modulation agent is one or more members selected from
the group consisting of biocompatible phosphorescent molecules.
12. The pharmaceutical composition of claim 9, wherein the at least
one energy modulation agent is one or more members selected from
the group consisting of biocompatible phosphorescent molecules.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/603,539, filed Jan. 23, 2015, and claims priority on U.S.
Provisional Application 61/930,717, filed Jan. 23, 2014. This
application is also related to U.S. application Ser. No.
11/935,655, filed Nov. 6, 2007; Ser. No. 12/389,946, filed Feb. 20,
2009; Ser. No. 12/401,478, filed Mar. 10, 2009; Ser. No.
12/417,779, filed Apr. 3, 2009; Ser. No. 12/764,184, filed Apr. 21,
2010; Ser. No. 12/843,188, filed Jul. 26, 2010; Ser. No.
13/054,279, which is a national stage application of
PCT/US2009/050514, filed Jul. 14, 2009, Ser. No. 13/739,398, filed
Jan. 11, 2013; and Ser. No. 13/739,414, filed Jan. 11, 2013, and to
U.S. Pat. No. 8,389,958, issued Mar. 5, 2012, and U.S. Pat. No.
8,383,836, issued Feb. 26, 2013, the contents of which are hereby
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of Invention
[0003] The present invention relates to methods for treating cell
proliferation disorders involving applying a psoralen derivative
lacking a DNA cross-linking motif to cancer cells, a psoralen or a
derivative thereof and lapatinib, or a psoralen or derivative
thereof and neratinib, to a subject and applying initiation
radiation energy from an energy source.
[0004] 2. Discussion of the Background
Cell Proliferation Disorders
[0005] There are several types of cell proliferation disorders.
Exemplary cell proliferation disorders may include, but are not
limited to, cancer, bacterial infection, immune rejection response
of organ transplant, solid tumors, viral infection, autoimmune
disorders (such as arthritis, lupus, inflammatory bowel disease,
Sjogrens syndrome, multiple sclerosis) or a combination thereof, as
well as aplastic conditions wherein cell proliferation is low
relative to healthy cells, such as aplastic anemia. Of these,
cancer is perhaps the most well known. The term "cancer" generally
refers to a diverse class of diseases that are commonly
characterized by an abnormal proliferation of the diseased cells. A
unifying thread in all known types of cancer is the acquisition of
abnormalities in the genetic material of the cancer cell and its
progeny. Once a cell becomes cancerous, it will proliferate without
respect to normal limits, invading and destroying adjacent tissues,
and may even spread to distant anatomic sites through a process
called metastasis. These life-threatening, malignant properties of
cancers differentiate them from benign tumors, which are
self-limited in their growth and do not invade or metastasize.
[0006] The impact of cancer on society cannot be overstated. The
disease may affect people at all ages, with a risk factor that
significantly increases with a person's age. It has been one of the
principal causes of death in developed countries and, as our
population continues to age, it is expected to be an even greater
threat to our society and economy. Therefore, finding cures and
effective treatments for cancer has been, and remains, a priority
within the biomedical research community.
Psoralens and Related Compounds
[0007] U.S. Pat. No. 6,235,508 further teaches that psoralens are
naturally occurring compounds which have been used therapeutically
for millennia in Asia and Africa. The action of psoralens and light
has been used to treat vitiligo and psoriasis (PUVA therapy;
Psoralen Ultra Violet A). Psoralen is capable of binding to nucleic
acid double helices by intercalation between base pairs; adenine,
guanine, cytosine and thymine (DNA) or uracil (RNA). Upon
sequential absorption of two UV-A photons, psoralen in its excited
state reacts with a thymine or uracil double bond and covalently
attaches to both strands of a nucleic acid helix. The crosslinking
reaction appears to be specific for a thymine (DNA) or a uracil
(RNA) base. Binding proceeds only if psoralen is intercalated in a
site containing thymine or uracil, but an initial photoadduct must
absorb a second UVA photon to react with a second thymine or uracil
on the opposing strand of the double helix in order to crosslink
each of the two strands of the double helix, as shown below. This
is a sequential absorption of two single photons as shown, as
opposed to simultaneous absorption of two or more photons.
##STR00001##
[0008] In addition, the reference teaches that 8-MOP is unsuitable
for use as an antiviral, because it damages both cells and viruses.
Lethal damage to a cell or virus occurs when the psoralen is
intercalated into a nucleic acid duplex in sites containing two
thymines (or uracils) on opposing strands but only when it
sequentially absorbs 2 UVA photons and thymines (or uracils) are
present. U.S. Pat. No. 4,748,120 of Wiesehan is an example of the
use of certain substituted psoralens by a photochemical
decontamination process for the treatment of blood or blood
products.
[0009] Additives, such as antioxidants are sometimes used with
psoralens, such as 8-MOP, AMT and I-IMT, to scavenge singlet oxygen
and other highly reactive oxygen species formed during
photoactivation of the psoralens. It is well known that UV
activation creates such reactive oxygen species, which are capable
of seriously damaging otherwise healthy cells. Much of the viral
deactivation may be the result of these reactive oxygen species
rather than any effect of photoactivation of psoralens. Regardless,
it is believed that no auto vaccine effect has been observed.
[0010] The best known photoactivatable compounds are derivatives of
psoralen or coumarin, which are nucleic acid intercalators. The use
of psoralen and coumarin photo sensitizers can give rise to
alternative chemical pathways for dissipation of the excited state
that are either not beneficial to the goal of viral inactivation,
or that are actually detrimental to the process. For psoralens and
coumarins, this chemical pathway is likely to lead to the formation
of a variety of ring-opened species, such as shown below for
coumarin:
##STR00002##
[0011] Research in this field over-simplifies mechanisms involved
in the photoactivating mechanism and formation of highly reactive
oxygen species, such as singlet oxygen. Both may lead to
inactivating damage of tumor cells, viruses and healthy cells.
However, neither, alone or combined, lead to an auto vaccine
effect. This requires an activation of the body's own immune system
to identify a malignant cell or virus as threat and to create an
immune response capable of lasting cytotoxic effects directed to
that threat. It is believed, without being limiting in any way,
that photoactivation and the resulting apoptosis of malignant cells
that occurs in extracorporeal photophoresis causes the activation
of an immune response with cytotoxic effects on untreated malignant
cells. While the complexity of the immune response and cytotoxid
effects is fully appreciated by researchers, a therapy that
harnesses the system to successfully stimulate an auto vaccine
effect against a targeted, malignant cell has been elusive, except
for extracorporeal photophoresis for treating lymphoma.
[0012] Midden (W. R. Midden, Psoralen DNA photobiology, Vol I1 (ed.
F. P. Gaspalloco) CRC press, pp. 1. (1988) has presented evidence
that psoralens photoreact with unsaturated lipids and photoreact
with molecular oxygen to produce active oxygen species such as
superoxide and singlet oxygen that cause lethal damage to
membranes.
[0013] U.S. Pat. No. 6,235,508 teaches that 8-MOP and AMT are
unacceptable photosensitizers, because each indiscriminately
damages both cells and viruses. Studies of the effects of cationic
side chains on furocoumarins as photosensitizers are reviewed in
Psoralen DNA Photobiology, Vol. I, ed. F. Gaspano, CRC Press, Inc.,
Boca Raton, Fla., Chapter 2. U.S. Pat. No. 6,235,508 gleans the
following from this review: most of the amino compounds had a much
lower ability to both bind and form crosslinks to DNA compared to
8-MOP, suggesting that the primary amino functionality is the
preferred ionic species for both photobinding and crosslinking.
[0014] U.S. Pat. No. 5,216,176 of Heindel discloses a large number
of psoralens and coumarins that have some effectiveness as
photoactivated inhibitors of epidermal growth factor. Halogens and
amines are included among the vast functionalities that could be
included in the psoralen/coumarin backbone. This reference is
incorporated herein by reference.
[0015] U.S. Pat. No. 5,984,887 discloses using extracorporeal
photophoresis with 8-MOP to treat blood infected with CMV. The
treated cells as well as killed and/or attenuated virus, peptides,
native subunits of the virus itself (which are released upon cell
break-up and/or shed into the blood) and/or pathogenic
noninfectious viruses are then used to generate an immune response
against the virus, which was not present prior to the
treatment.
Treatment Methods
[0016] Existing treatments for cell proliferation disorders such as
cancer include surgery, chemotherapy, radiation therapy,
immunotherapy, monoclonal antibody therapy, and several other
lesser known methods. The choice of therapy usually depends on the
location and severity of the disorder, the stage of the disease, as
well as the patient's response to the treatment.
[0017] While some treatments may only seek to manage and alleviate
symptoms of the disorder, the ultimate goal of any effective
therapy is the complete removal or cure of all disordered cells
without damage to the rest of the body. With cancer, although
surgery may sometimes accomplish this goal, the propensity of
cancer cells to invade adjacent tissue or to spread to distant
sites by microscopic metastasis often limits the effectiveness of
this option. Similarly, the effectiveness of current chemotherapy
is often limited by toxicity to other tissues in the body.
Radiation therapy suffers from similar shortcomings as other
aforementioned treatment methods. Most of these cancer treatment
methods, including radiation therapy, are known to cause damage to
DNA, which if not repaired during a critical stage in mitosis, the
splitting of the cell during cell proliferation, leads to a
programmed cell death, i.e. apoptosis. Further, radiation tends to
damage healthy cells, as well as malignant tumor cells.
[0018] A number of patents describe ex vivo treatment of bodily
fluids, for example blood. Blood is obtained from a patient,
treated with a photosensitive agent, exposed to UV radiation, and
reinjected to the patient (i.e. extracorporeal photopheresis).
Alternatively, a patient can be treated in vivo with a
photosensitive agent followed by the withdrawal of a sample from
the patient, treatment with UV radiation in vitro (ex vivo), and
reinjecting the patient with the treated sample. This method is
known for producing an autovaccine. A method of treating a patient
with a photosensitive agent, exposing the patient to an energy
source and generating an autovaccine effect wherein all steps are
conducted in vivo has not been described. See WO 03/049801, U.S.
Pat. No. 6,569,467; U.S. Pat. No. 6,204,058; U.S. Pat. No.
5,980,954; U.S. Pat. No. 6,669,965; U.S. Pat. No. 4,838,852; U.S.
Pat. No. 7,045,124, and U.S. Pat. No. 6,849,058. Moreover, the side
effects of extracorporeal photopheresis are well known and include
nausea, vomiting, cutaneous erythema, hypersensitivity to sunlight,
and secondary hematologic malignancy. Researchers are attempting to
use photopheresis in experimental treatments for patients with
cardiac, pulmonary and renal allograft rejection; autoimmune
diseases, and ulcerative colitis.
[0019] A survey of known treatment methods reveals that these
methods tend to face a primary difficulty of differentiating
between normal cells and target cells when delivering treatment,
often due to the production of singlet oxygen which is known to be
non-selective in its attack of cells, as well as the need to
perform the processes ex vivo, or through highly invasive
procedures, such as surgical procedures in order to reach tissues
more than a few centimeters deep within the subject.
[0020] U.S. Pat. No. 5,829,448 describes simultaneous two photon
excitation of photo-agents using irradiation with low energy
photons such as infrared or near infrared light (NRI). A single
photon and simultaneous two photon excitation is compared for
psoralen derivatives, wherein cells are treated with the photo
agent and are irradiated with NRI or UV radiation. The patent
suggests that treating with a low energy irradiation is
advantageous because it is absorbed and scattered to a lesser
extent than UV radiation. However, the use of NRI or UV radiation
is known to penetrate tissue to only a depth of a few centimeters.
Thus any treatment deep within the subject would necessarily
require the use of ex vivo methods or highly invasive techniques to
allow the irradiation source to reach the tissue of interest.
[0021] Chen et al., J. Nanosci. and Nanotech., 6:1159-1166 (2006);
Kim et al., JACS, 129:2669-2675 (2007); U.S. 2002/0127224; and U.S.
Pat. No. 4,979,935 each describe methods for treatment using
various types of energy activation of agents within a subject.
However, each suffers from the drawback that the treatment is
dependent on the production of singlet oxygen to produce the
desired effect on the tissue being treated, and is thus largely
indiscriminate in affecting both healthy cells and the diseased
tissue desired to be treated.
[0022] U.S. Pat. No. 6,908,591 discloses methods for sterilizing
tissue with irradiation to reduce the level of one or more active
biological contaminants or pathogens, such as viruses, bacteria,
yeasts, molds, fungi, spores, prions or similar agents responsible,
alone or in combination, for transmissible spongiform
encephalopathies and/or single or multicellular parasites, such
that the tissue may subsequently be used in transplantation to
replace diseased and/or otherwise defective tissue in an animal.
The method may include the use of a sensitizer such as psoralen, a
psoralen-derivative or other photosensitizer in order to improve
the effectiveness of the irradiation or to reduce the exposure
necessary to sterilize the tissue. However, the method is not
suitable for treating a patient and does not teach any mechanisms
for stimulating the photosensitizers, indirectly.
[0023] U.S. Pat. No. 6,235,508 discloses antiviral applications for
psoralens and other photoactivatable molecules. It teaches a method
for inactivating viral and bacterial contaminants from a biological
solution. The method includes mixing blood with a photosensitizer
and a blocking agent and irradiating the mixture to stimulate the
photosensitizer, inactivating substantially all of the contaminants
in the blood, without destroying the red blood cells. The blocking
agent prevents or reduces deleterious side reactions of the
photosensitizer, which would occur if not in the presence of the
blocking agent. The mode of action of the blocking agent is not
predominantly in the quenching of any reactive oxygen species,
according to the reference.
[0024] Also, U.S. Pat. No. 6,235,508 suggests that halogenated
photosensitizers and blocking agents might be suitable for
replacing 8-methoxypsoralen (8-MOP) in photophoresis and in
treatment of certain proliferative cancers, especially solid
localized tumors accessible via a fiber optic light device or
superficial skin cancers. However, the reference fails to address
any specific molecules for use in treating lymphomas or any other
cancer. Instead, the reference suggests a process of photophoresis
for antiviral treatments of raw blood and plasma.
[0025] U.S. Pat. No. 6,235,508 teaches away from 8-MOP and
4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) and many other
photoactivatable molecules, which are taught to have certain
disadvantages. Fluorescing photosensitizers are said to be
preferred, but the reference does not teach how to select a system
of fluorescent stimulation or photoactivation using fluorescent
photosensitizers. Instead, the fluorescing photosensitizer is
limited to the intercalator that is binding to the DNA. The
reference suggests that fluorescence indicates that such an
intercalator is less likely to stimulate oxygen radicals. Thus, the
reference fails to disclose any mechanism of photoactivation of an
intercalator other than by direct photoactivation by UV light,
although use of a UV light probe or X-rays is suggested for
penetrating deeper into tissues. No examples are provided for the
use of a UV light probe or for use of X-rays. No example of any
stimulation by X-ray radiation is taught.
PUVA
[0026] Methoxypsoralen (8MOP) is a linear tricyclic molecule that
readily enters cell nuclei where it intercalates DNA at
pyrimidine-purine sites [1]. Following photo-activation by UV
irradiation, a combination referred to as PUVA, 8MOP interacts with
pyrimidines to form stable DNA monoadducts. Upon further UVA
treatment, a percentage of monoadducts can then be converted to
interstrand DNA crosslinks (ICL), which in turn inhibit
transcription and DNA replication [1,2]. Importantly, the
anti-proliferative effects of PUVA appear to be related to the
formation of ICL, rather than monoadducts. Because of its
anti-proliferative effects, PUVA has been used to treat
hyperproliferative skin conditions including psoriasis [3].
Furthermore, T lymphocytes--normal and malignant--appear to be
particularly sensitive to the anti-proliferative effects of PUVA
therapy; hence, the use of PUVA as a treatment for cutaneous T-cell
lymphoma and graft-versus-host disease [4-6].
[0027] In addition to playing a role in the formation of ICL, there
is evidence that psoralen may also target non-nuclear proteins,
lipids, and cellular membrane components [7-9]. For example, Laskin
et al used psoralen derivatives incapable of forming DNA adducts in
response to UV irradiation to show that PUVA treatment blocked the
mitogenic effects of soluble Epidermal Growth Factor (EGF) on its
cognate cell surface receptor, EGF Receptor (EGFR) [7,9].
Interestingly, inhibition of EGFR phosphorylation in response to
PUVA was not mediated through a direct psoralen-EGFR interaction,
but rather psoralen interacting with a lower molecular weight
binding protein.
[0028] ErbB family receptors are Class I receptor tyrosine kinases
(Grassot J, Mouchiroud G, Peniere G., RTKdb: database of Receptor
Tyrosine Kinase, Nucleic Acids Res., 31(1): 353-8 (2003))). ERBB2
(also known as HER-2 or NEU) appears to act as an essential partner
for the other members of the family without itself being activated
by a cognate ligand (Graus-Porta D, Beerli R R, Daly J M, Hynes N
E, ErbB-2, the preferred heterodimerization partner of all ErbB
receptors, is a mediator of lateral signaling, EMBO J.,
16(7):1647-55 (1997)). Ligands of the ErbB family of receptors are
peptides, many of which are generated by proteolytic cleavage of
cell-surface proteins. HER/ErbB is the viral counterpart to the
receptor tyrosine kinase EGFR. All family members heterodimerize
with each other to activate downstream signaling pathways and are
aberrantly expressed in many cancers, particularly forms of breast
cancer.
[0029] Similar to EGFR, the ErbB2 oncogene is a member of the type
1 transmembrane family of receptor tyrosine kinases. Gene
amplification and overexpression of ErbB2, which occurs in 25% of
all breast cancers, predicts for a poor clinical outcome as a
consequence of increased tendency to metastasize to visceral organs
earlier in the disease course [10,11]. These findings have prompted
the development of ErbB2 targeted therapies--biological and small
molecule tyrosine kinase inhibitors (TKIs)--for the treatment of
early and advanced stage ErbB2+ breast cancers [12]. Although ErbB2
targeted therapies represent a significant advancement in the
treatment of aggressive breast cancers, their clinical efficacy has
been limited by the inevitable development of therapeutic
resistance, particularly in the advanced stage setting [13-15].
[0030] Using mass spectroscopy and biochemical approaches, the
inventors have now shown for the first time that photo-activated
8MOP can directly interact with regulatory elements within the
ErbB2 catalytic kinase domain, providing a likely explanation for
the targeted inhibition of ErbB2 signaling in response to PUVA
therapy. Furthermore, a modified psoralen derivative that lacks the
ability to crosslink DNA maintained its ability to block ErbB2
signaling and induce tumor cell apoptosis. The inventors have also
shown that PUVA can trigger significant apoptosis in ErbB2+ breast
cancer models of acquired therapeutic resistance to lapatinib and
similar ErbB2 targeted therapies. These findings affect the
development of new therapeutic strategies for ErbB+ breast cancers,
including those that have become resistant to existing ErbB2
targeted therapies.
SUMMARY OF THE INVENTION
[0031] Accordingly, one object of the present invention is to
provide a method for treatment of a cell proliferation disease or
disorder with a psoralen derivative and combination therapy.
[0032] A further object of the present invention is to provide a
method of treating a cell proliferation disorder or disease with a
psoralen derivative lacking a DNA cross-linking motif and radiation
energy.
[0033] A further object of the present invention is to provide a
method of treating a cell proliferation disorder or disease with a
psoralen or psoralen derivative, lapatinib and radiation
energy.
[0034] A further object of the present invention is to provide a
method of treating a cell proliferation disorder or disease with a
psoralen or psoralen derivative, neratinib and radiation
energy.
[0035] A still further object of the present invention is to
provide a method of inhibition of ErbB2 signaling in cancer cells
with a psoralen derivative lacking a DNA cross-linking motif and
radiation energy.
[0036] These and other objects of the present invention, which will
become more apparent in conjunction with the following detailed
description of the preferred embodiments, either alone or in
combinations thereof, have been satisfied by the discovery of a
method of treating a cell proliferation disorder or disease
comprising administering a psoralen derivative lacking a DNA
cross-linking motif or a combination of a psoralen or its
derivative and lapatinib or neratinib to a subject in need thereof
and applying initiation radiation energy form an energy source.
BRIEF DESCRIPTION OF THE FIGURES
[0037] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the office
upon request and payment of the necessary fee.
[0038] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0039] FIG. 1 A-F show PUVA antitumor activity in HER2+ breast
cancer cells. Cells were pre-treated with the indicated
concentrations of 8MOP for 4 hr before UVA irradiation (2J), and
then cultured for an additional 72 hr before being analyzed for
cell growth (A) BT474; (B) SKBR3; (C) MCF7, (D) HFF and apoptosis
(E) BT474; (F) SKBR3. Cells treated with vehicle (0.01% DMSO) alone
served as controls. Results represent the mean+/- standard error of
triplicate samples, and are representative of three independent
experiments.
[0040] FIG. 2 shows that PUVA therapy inhibits ErbB2 signaling.
BT474, SKBR3 and MCF7 cells were subjected to the indicated
treatment conditions as described in FIG. 1. Western blot analysis
was performed on whole cell lysates. Actin steady-state protein
levels served as a control to ensure for equal loading of protein.
Results are representative of three independent experiments.
[0041] FIG. 3 A-C show inhibition of ErbB2 signaling in response to
PUVA is independent of DNA crosslinking. The chemical structures of
(A) 8MOP, and (B) 7-methylpyridopsoralen (SMSF032310), which is a
derivative of 8MOP that lacks the ability to crosslink DNA, are
shown. (C) BT474 and SKBR3 cells were exposed to the indicated
treatments. Cell growth and viability assays were performed after
72 hr. P<0.0003 (SKBR3); P<0.0009 (BT474). Cells treated with
vehicle alone served as controls. Results represent the mean+/-
standard error of triplicate samples, and are representative of
three independent experiments. Corresponding Western blot analysis
of the indicated protein/phosphoproteins is shown. Steady-state
actin protein levels served as controls for equal loading of
proteins. Results are representative of three independent
experiments.
[0042] FIG. 4 A-B show that 8MOP interacts with the catalytic
kinase domain of ErbB2. (A) 8MOP interacts with three peptide
regions within the ErbB2 catalytic kinase domain. Qualitative
peptide identifications within the ErbB2 catalytic kinase domain
following LC-MS/MS analysis of a streptavidin pull-down of
biotinylated-8MOP bait (see Material and Methods). The
transmembrane domain is indicated (red diamond) and the five
C-terminus tyrosine autophosphorylation sites are indicated (p).
(B) Non-reducing Western blot analysis of the interaction of 8MOP
with ErbB2. BT474 cells were treated with 800dye-8MOP (Promega) or
with vehicle (0.01% DMSO) alone served as control for 48 hr and
then exposed to UV irradiation (2J) prior to Western blot analysis.
The image on the left shows the Western blot for ErbB2 (red). The
image on the right shows the same membrane directly scanned for the
presence of 800dye-8MOP (green), which overlays the ErbB2 signal.
The results are representative of three independent
experiments.
[0043] FIG. 5 The combination of PUVA with the irreversible
pan-ErbB TKI neratinib results in enhanced antitumor activity. The
growth and viability of BT474 and SKBR3 cells (top bar graphs)
after being subjected to the indicated treatment conditions. The
combination of PUVA plus neratinib: P<0.0005 (BT474 and SKBR3
cells). Results represent the mean+/- standard error of triplicate
samples, and are representative of three independent experiments.
(B) Western blot analysis showing steady-state ErbB2, ErbB3, and
phospho-Akt (S473) protein levels in BT474 and SKBR3 cells treated
according to the indicated treatment conditions. Vehicle alone
(0.01% DMSO) served as a control. Steady-state actin protein levels
served as a control for equal loading of protein. The results are
representative of three independent experiments.
[0044] FIG. 6 A-B shows that PUVA therapy reverses acquired
resistant to ErbB2 targeted therapies. (A) Equal numbers of rBT474
and rSKBR3 cells were subjected to the indicated treatment
conditions, and the effects on cell growth and viability are shown.
P values of statistical significance are indicated. Results
represent the mean+/- standard error of triplicate samples, and are
representative of three independent experiments. (B) The
corresponding Western blot analysis for each of the indicated
treatment conditions is shown. As indicated, rBT474 and rSKBR3
cells are continuously maintained in 1 .mu.M lapatinib. Actin
steady-state protein levels served as a control to ensure for equal
loading of protein. Results are representative of three independent
experiments.
[0045] FIG. 7 PUVA therapy targets nuclear p85.sup.ErbB2, inducing
tumor cell apoptosis. Top bar graph shows the results of the growth
assays performed in T47D and stably transfected T47D cell line.
T47D cells expressing p85.sup.ErbB2 were pretreated with 5 .mu.M
lapatinib or 5 .mu.M 8MOP for 4 hr followed by irradiation in a UV
Stratalinker 1800 (Statagene). Cells transfected with empty vector
(T47D/Vector), and those treated with vehicle alone (0.01% DMSO)
served as controls. The effects of the treatments on cell growth
and viability are shown in the bar graph. P<0.0071 (8MOP+UVA
irradiation). Results represent the mean+/- standard error of
triplicate samples, and are representative of three independent
experiments. Steady-state phospho-p85.sup.ErbB2 protein levels
(dotted arrow) and phospho-p185.sup.ErbB2 (solid arrow) are shown
by Western blot. Actin steady-state protein levels served as a
control to ensure for equal loading of protein. Results are
representative of three independent experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0046] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. Further, the materials, methods, and examples are
illustrative only and are not intended to be limiting, unless
otherwise specified.
[0047] The present invention sets forth novel methods of treating
cell proliferation disorders that is effective, specific, and has
few side-effects. Those cells suffering from a cell proliferation
disorder are referred to herein as the target cells. A treatment
for cell proliferation disorders, including solid tumors, is
capable of chemically binding cellular nucleic acids, including but
not limited to, the DNA or mitochondrial DNA or RNA of the target
cells. For example, a photoactivatable agent, such as a psoralen or
a psoralen derivative, is exposed in situ to an energy source
capable of activating the photoactivatable agent or agents
selected. In another example, the photoactivatable agent is a
photosensitizer. The photoactivatable agent may be a metal
nanocluster or a molecule.
[0048] As noted above, an object of the present invention is to
treat cell proliferation disorders. Exemplary cell proliferation
disorders may include, but are not limited to, cancer, as well as
bacterial and viral infections where the invading bacteria grows at
a much more rapid rate than cells of the infected host. In
addition, treatment for certain developmental stage diseases
related to cell proliferation, such as syndactyly, are also
contemplated.
[0049] Accordingly, in one embodiment, the present invention
provides methods that are capable of overcoming the shortcomings of
the existing methods. In general, a method in accordance with the
present invention utilizes the principle of energy transfer to and
among molecular agents to control delivery and activation of
pharmaceutically active agents such that delivery of the desired
pharmacological effect is more focused, precise, and effective than
the conventional techniques.
[0050] Although not intending to be bound by any particular theory
or be otherwise limited in any way, the following theoretical
discussion of scientific principles and definitions are provided to
help the reader gain an understanding and appreciation of the
present invention.
[0051] As used herein, the term "subject" is not intended to be
limited to humans, but may also include animals, plants, or any
suitable biological organism.
[0052] As used herein, the phrase "cell proliferation disorder"
refers to any condition where the growth rate of a population of
cells is less than or greater than a desired rate under a given
physiological state and conditions. Although, preferably, the
proliferation rate that would be of interest for treatment purposes
is faster than a desired rate, slower than desired rate conditions
may also be treated by methods of the present invention. Exemplary
cell proliferation disorders may include, but are not limited to,
cancer, bacterial infection, immune rejection response of organ
transplant, solid tumors, viral infection, autoimmune disorders
(such as arthritis, lupus, inflammatory bowel disease, Sjogrens
syndrome, multiple sclerosis) or a combination thereof, as well as
aplastic conditions wherein cell proliferation is low relative to
healthy cells, such as aplastic anemia. Particularly preferred cell
proliferation disorders for treatment using the present methods are
cancer, staphylococcus aureus (particularly antibiotic resistant
strains such as methicillin resistant staphylococcus aureus or
MRSA), and autoimmune disorders.
[0053] As used herein, an "activatable pharmaceutical agent" is an
agent that normally exists in an inactive state in the absence of
an activation signal. When the agent is activated by a matching
activation signal under activating conditions, it is capable of
effecting the desired pharmacological effect on a target cell (i.e.
preferably a predetermined cellular change).
[0054] Signals that may be used to activate a corresponding agent
may include, but are not limited to, photons of specific
wavelengths (e.g. x-rays, or visible light), electromagnetic energy
(e.g. radio or microwave), thermal energy, acoustic energy, or any
combination thereof.
[0055] Activation of the agent may be as simple as delivering the
signal to the agent or may further premise on a set of activation
conditions. For example, in the former case, an activatable
pharmaceutical agent, such as a photosensitizer, may be activated
by UV-A radiation. Once activated, the agent in its active-state
may then directly proceed to effect a cellular change.
[0056] Where activation may further premise upon other conditions,
mere delivery of the activation signal may not be sufficient to
bring about the desired cellular change. For example, a photoactive
compound that achieves its pharmaceutical effect by binding to
certain cellular structure in its active state may require physical
proximity to the target cellular structure when the activation
signal is delivered. For such activatable agents, delivery of the
activation signal under non-activating conditions will not result
in the desired pharmacologic effect. Some examples of activating
conditions may include, but are not limited to, temperature, pH,
location, state of the cell, presence or absence of co-factors.
[0057] Selection of an activatable pharmaceutical agent greatly
depends on a number of factors such as the desired cellular change,
the desired form of activation, as well as the physical and
biochemical constraints that may apply. Exemplary activatable
pharmaceutical agents may include, but are not limited to, agents
that may be activated by photonic energy, electromagnetic energy,
acoustic energy, chemical or enzymatic reactions, thermal energy,
or any other suitable activation mechanisms.
[0058] When activated, the activatable pharmaceutical agent may
effect cellular changes that include, but are not limited to,
apoptosis, redirection of metabolic pathways, up-regulation of
certain genes, down-regulation of certain genes, secretion of
cytokines, alteration of cytokine receptor responses, or
combinations thereof.
[0059] The mechanisms by which an activatable pharmaceutical agent
may achieve its desired effect are not particularly limited. Such
mechanisms may include direct action on a predetermined target as
well as indirect actions via alterations to the biochemical
pathways. A preferred direct action mechanism is by binding the
agent to a critical cellular structure such as nuclear DNA, mRNA,
rRNA, ribosome, mitochondrial DNA, or any other functionally
important structures. Indirect mechanisms may include releasing
metabolites upon activation to interfere with normal metabolic
pathways, releasing chemical signals (e.g. agonists or antagonists)
upon activation to alter the targeted cellular response, and other
suitable biochemical or metabolic alterations.
[0060] In one preferred embodiment, the activatable pharmaceutical
agent is capable of chemically binding to the DNA or mitochondria
at a therapeutically effective amount. In this embodiment, the
activatable pharmaceutical agent, preferably a photoactivatable
agent, is exposed in situ to an activating energy emitted from an
energy modulation agent, which, in turn receives energy from an
initiation energy source.
[0061] Suitable activatable agents include, but are not limited to,
photoactive agents, sono-active agents, thermo-active agents, and
radio/microwave-active agents. An activatable agent may be a small
molecule; a biological molecule such as a protein, a nucleic acid
or lipid; a supramolecular assembly; a nanoparticle; or any other
molecular entity having a pharmaceutical activity once
activated.
[0062] The activatable agent may be derived from a natural or
synthetic origin. Any such molecular entity that may be activated
by a suitable activation signal source to effect a predetermined
cellular change may be advantageously employed in the present
invention.
[0063] Suitable photoactive agents include, but are not limited to:
psoralens and psoralen derivatives, pyrene cholesteryloleate,
acridine, porphyrin, fluorescein, rhodamine, 16-diazorcortisone,
ethidium, transition metal complexes of bleomycin, transition metal
complexes of deglycobleomycin, organoplatinum complexes,
alloxazines such as 7,8-dimethyl-10-ribityl isoalloxazine
(riboflavin), 7,8,10-trimethylisoalloxazine (lumiflavin),
7,8-dimethylalloxazine (lumichrome), isoalloxazine-adenine
dinucleotide (flavine adenine dinucleotide [FAD]), alloxazine
mononucleotide (also known as flavine mononucleotide [FMN] and
riboflavine-5-phosphate), vitamin Ks, vitamin L, their metabolites
and precursors, and napththoquinones, naphthalenes, naphthols and
their derivatives having planar molecular conformations,
porphyrins, dyes such as neutral red, methylene blue, acridine,
toluidines, flavine (acriflavine hydrochloride) and phenothiazine
derivatives, coumarins, quinolones, quinones, and anthroquinones,
aluminum (111) phthalocyanine tetrasulfonate, hematoporphyrin, and
phthalocyanine, and compounds which preferentially adsorb to
nucleic acids with little or no effect on proteins. The term
"alloxazine" includes isoalloxazines.
[0064] Endogenously-based derivatives include synthetically derived
analogs and homologs of endogenous photoactivated molecules, which
may have or lack lower (1 to 5 carbons) alkyl or halogen
substituents of the photosensitizers from which they are derived,
and which preserve the function and substantial non-toxicity.
Endogenous molecules are inherently non-toxic and may not yield
toxic photoproducts after photoradiation.
[0065] The nature of the predetermined cellular change will depend
on the desired pharmaceutical outcome. Exemplary cellular changes
may include, but are not limited to, apoptosis, necrosis,
up-regulation of certain genes, down-regulation of certain genes,
secretion of cytokines, alteration of cytokine receptor responses,
or a combination thereof.
[0066] As used herein, an "energy modulation agent" refers to an
agent that is capable of receiving an energy input from a source
and then re-emitting a different energy to a receiving target.
Energy transfer among molecules may occur in a number of ways. The
form of energy may be electronic, thermal, electromagnetic,
kinetic, or chemical in nature. Energy may be transferred from one
molecule to another (intermolecular transfer) or from one part of a
molecule to another part of the same molecule (intramolecular
transfer). For example, a modulation agent may receive
electromagnetic energy and re-emit the energy in the form of
thermal energy. In preferred embodiments, the energy modulation
agent receives higher energy (e.g. x-ray) and re-emits in lower
energy (e.g. UV-A). Some modulation agents may have a very short
energy retention time (on the order of fs, e.g. fluorescent
molecules) whereas others may have a very long half-life (on the
order of minutes to hours, e.g. luminescent or phosphorescent
molecules). Suitable energy modulation agents include, but are not
limited to, a biocompatible fluorescing metal nanoparticle,
fluorescing dye molecule, gold nanoparticle, a water soluble
quantum dot encapsulated by polyamidoamine dendrimers, a
luciferase, a biocompatible phosphorescent molecule, a combined
electromagnetic energy harvester molecule, and a lanthanide chelate
capable of intense luminescence. Various exemplary uses of these
are described below in preferred embodiments.
[0067] The modulation agents may further be coupled to a carrier
for cellular targeting purposes. For example, a biocompatible
molecule, such as a fluorescing metal nanoparticle or fluorescing
dye molecule that emits in the UV-A band, may be selected as the
energy modulation agent.
[0068] The energy modulation agent may be preferably directed to
the desired site (e.g. a tumor) by systemic administration to a
subject. For example, a UV-A emitting energy modulation agent may
be concentrated in the tumor site by physical insertion or by
conjugating the UV-A emitting energy modulation agent with a tumor
specific carrier, such as a lipid, chitin or chitin-derivative, a
chelate or other functionalized carrier that is capable of
concentrating the UV-A emitting source in a specific target
tumor.
[0069] Additionally, the energy modulation agent can be used alone
or as a series of two or more energy modulation agents wherein the
energy modulation agents provide an energy cascade. Thus, the first
energy modulation agent in the cascade will absorb the activation
energy, convert it to a different energy which is then absorbed by
the second energy modulation in the cascade, and so forth until the
end of the cascade is reached with the final energy modulation
agent in the cascade emitting the energy necessary to activate the
activatable pharmaceutical agent.
[0070] Although the activatable pharmaceutical agent and the energy
modulation agent can be distinct and separate, it will be
understood that the two agents need not be independent and separate
entities. In fact, the two agents may be associated with each other
via a number of different configurations. Where the two agents are
independent and separately movable from each other, they generally
interact with each other via diffusion and chance encounters within
a common surrounding medium. Where the activatable pharmaceutical
agent and the energy modulation agent are not separate, they may be
combined into one single entity.
[0071] The energy emitting source can be any energy source capable
of providing energy at a level sufficient to activate the
activatable agent directly, or to provide the energy modulation
agent with the input needed to emit the activation energy for the
activatable agent (indirect activation). Preferable initiation
energy sources include, but are not limited to, UV-A lamps or fiber
optic lines, a light needle, an endoscope, and a linear accelerator
that generates x-ray, gamma-ray, or electron beams. In a preferred
embodiment the initiation energy source is a source of energy
capable of penetrating completely through the subject. Within the
context of the present invention, the phrase "capable of
penetrating completely through the subject" is used to refer to
sources of energy that can penetrate to any depth within the
subject to activate the activatable pharmaceutical agent. It is not
required that the any of the energy applied actually pass
completely through the subject, merely that it be capable of doing
so in order to permit penetration to any desired depth to activate
the activatable pharmaceutical agent. Exemplary initiation energy
sources that are capable of penetrating completely through the
subject include, but are not limited to, x-rays, gamma rays,
electron beams, microwaves and radio waves.
[0072] Alternatively, the energy emitting source may be another
energy modulation agent that emits energy in a form suitable for
absorption by the transfer agent. For example, the initiation
energy source may be acoustic energy and one energy modulation
agent may be capable of receiving acoustic energy and emitting
photonic energy (e.g. sonoluminescent molecules) to be received by
another energy modulation agent that is capable of receiving
photonic energy. Other examples include transfer agents that
receive energy at x-ray wavelength and emit energy at UV
wavelength, preferably at UV-A wavelength. As noted above, a
plurality of such energy modulation agents may be used to form a
cascade to transfer energy from initiation energy source via a
series of energy modulation agents to activate the activatable
agent.
[0073] Signal transduction schemes as a drug delivery vehicle may
be advantageously developed by careful modeling of the cascade
events coupled with metabolic pathway knowledge to sequentially or
simultaneously activate multiple activatable pharmaceutical agents
to achieve multiple-point alterations in cellular function.
[0074] Photoactivatable agents may be stimulated by an energy
source, such as irradiation, resonance energy transfer, exciton
migration, electron injection, or chemical reaction, to an
activated energy state that is capable of effecting the
predetermined cellular change desired. In a preferred embodiment,
the photoactivatable agent, upon activation, binds to DNA or RNA or
other structures in a cell. The activated energy state of the agent
is capable of causing damage to cells, inducing apoptosis. The
mechanism of apoptosis is associated with an enhanced immune
response that reduces the growth rate of cell proliferation
disorders and may shrink solid tumors, depending on the state of
the patient's immune system, concentration of the agent in the
tumor, sensitivity of the agent to stimulation, and length of
stimulation.
[0075] Ultraviolet A activation of psoralen, a therapy referred to
as PUVA, is an effective treatment for non-malignant and malignant
proliferative skin disorders. The mechanism of action has been
attributed to psoralen intercalation of DNA, which upon UV
treatment, leads to formation of interstrand DNA crosslinks (ICL),
and induction of cell apoptosis. Here, the inventors have
discovered a new mechanism of action of PUVA in models of breast
cancer that overexpress the ErbB2 receptor tyrosine kinase
oncogene. PUVA blocked tyrosine autophosphorylation/activation of
ErbB2 with concomitant inhibition of downstream PI3K and MAPK
signaling pathways, triggering tumor cell apoptosis. Importantly,
photo-activation of a modified psoralen derivative,
7-methylpyridopsoralen (SMSF032310)
##STR00003##
[0076] that lacks the DNA cross-linking motif retained the ability
to block ErbB2 signaling and induce tumor cell death. Using a mass
spectroscopy-based platform, the inventors have shown that 8-MOP
(8-methoxypsoralen) can interact with the catalytic kinase domain
of ErbB2. Importantly, the antitumor effects of PUVA do not appear
to be cross resistant with other ErbB2 targeted therapies, as PUVA
can induce apoptosis in established ErbB2+ cancer models of
acquired lapatinib resistance. Thus, PUVA represents a novel ErbB2
targeted therapy for the treatment of ErbB2+ breast cancers,
including those that have developed resistance to other ErbB2
targeted therapies.
[0077] A preferred method of inhibition of ErbB2 signaling in
cancer cells comprises applying a psoralen derivative lacking a DNA
cross-linking motif to cancer cells and applying initiation
radiation energy form an energy source, thereby blocking the ErbB2
signaling. In one embodiment the cancer cells may be ErbB2+ breast
cancer cells.
[0078] The psoralen derivative lacking a DNA cross-linking motif is
preferably a psoralen derivative of Formula (I):
##STR00004##
[0079] wherein R.sub.1 is hydrogen, lower alkyl, or lower
alkoxy;
[0080] R.sub.2 and R.sub.3 are each, independently, hydrogen,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy, or R.sub.2 and
R.sub.3 may join to form a substituted or unsubstituted, condensed
5 to 7 membered aliphatic or aromatic ring, optionally containing a
heteroatom selected from N, S, or O;
[0081] R.sub.4 and R.sub.5 are each, independently, hydrogen,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkoxy, or R.sub.4 and
R.sub.5 may join to form a substituted or unsubstituted, condensed
5 to 7 membered aliphatic or aromatic ring, optionally containing a
heteroatom selected from N, S, or O;
[0082] With the proviso that at least one of R.sub.2 and R.sub.3 or
R.sub.4 and R.sub.5 must be substituents sufficient to prevent the
formation of DNA cross-links, preferably at least one of R.sub.2
and R.sub.3 or R.sub.4 and R.sub.5 join to form a substituted or
unsubstituted, condensed 5 to 7 membered aliphatic or aromatic
ring, optionally containing a heteroatom selected from N, S, or
O.
[0083] In a preferred embodiment, R.sub.2 and R.sub.3 or R.sub.4
and R.sub.5 join to form a condensed aromatic heterocycle, most
preferably a pyridyl ring. A most preferred embodiment of compound
in which DNA crosslinking is blocked is 7-methylpyridopsoralen
having the following structure:
##STR00005##
[0084] In another embodiment, prior to applying the initiation
energy, at least one energy modulation agent that converts the
initiation energy to an energy that activates the psoralen
derivative, is administered to the subject.
[0085] In yet another embodiment, the energy modulation agent may
be one or more selected from a biocompatible fluorescing metal
nanoparticle, fluorescing dye molecule, gold nanoparticle, a water
soluble quantum dot encapsulated by polyamidoamine dendrimers, a
luciferase, a biocompatible phosphorescent molecule, a combined
electromagnetic energy harvester molecule, and a lanthanide chelate
capable of intense luminescence.
[0086] A preferred method of treating a cell proliferation disorder
or disease comprises administering a psoralen derivative lacking a
DNA cross-linking motif to a subject in need thereof and applying
radiation energy form an energy source, wherein the treatment is
caused by inducing apoptosis in diseased cells, thereby blocking
ErbB2 signaling in cancer cells. The cell proliferation disorder or
disease may be cancer and preferably, breast cancer. In one
embodiment, the diseased cells may be ErbB2+ breast cancer
cells.
[0087] In another embodiment, the radiation energy is UVA or
visible energy which may be applied directly or indirectly. The
initiation energy may be applied via a thin fiber optic.
[0088] In another embodiment, prior to said applying the initiation
energy, at least one energy modulation agent that converts the
initiation energy to an energy that activates the psoralen
derivative may be administered. The energy modulation agent may be
one or more selected from a biocompatible fluorescing metal
nanoparticle, fluorescing dye molecule, gold nanoparticle, a water
soluble quantum dot encapsulated by polyamidoamine dendrimers, a
luciferase, a biocompatible phosphorescent molecule, a combined
electromagnetic energy harvester molecule, and a lanthanide chelate
capable of intense luminescence.
[0089] Another preferred method of treating a cell proliferation
disorder or disease comprises administering a psoralen or psoralen
derivative and lapatinib to a subject in need thereof and applying
radiation energy form an energy source, wherein the treatment
reduces diseased cell growth and/or viability compared to that of
diseased cells treated with lapatinib alone, or a combination of
lapatinib and the psoralen or psoralen derivative, or a combination
of lapatinib and the radiation energy. In one embodiment, the
treatment reduces tumor cell growth and/or viability in lapatinib
resistant tumor cells.
[0090] The cell proliferation disorder or disease may be breast
cancer. In another embodiment, the lapatinib resistant tumor cells
may be HER2+ breast cancer cells.
[0091] In yet another embodiment, the radiation energy is UVA or
visible energy which may be applied directly or indirectly.
[0092] In another embodiment, a psoralen or psoralen derivative is
8-Methoxypsoralen (8-MOP).
[0093] In a different embodiment, prior to said applying the
initiation energy, at least one energy modulation agent that
converts the initiation energy to an energy that activates the
psoralen derivative may be administered to a subject. The energy
modulation agent may be one or more selected from a biocompatible
fluorescing metal nanoparticle, fluorescing dye molecule, gold
nanoparticle, a water soluble quantum dot encapsulated by
polyamidoamine dendrimers, a luciferase, a biocompatible
phosphorescent molecule, a combined electromagnetic energy
harvester molecule, and a lanthanide chelate capable of intense
luminescence.
[0094] Yet, another preferred method of treating a cell
proliferation disorder or disease comprises administering a
psoralen or psoralen derivative and neratinib to a subject in need
thereof and applying radiation energy form an energy source,
wherein the treatment reduces diseased cell growth and/or viability
compared to that of diseased cells treated with neratinib alone, or
a combination of neratinib and the psoralen or psoralen derivative,
or a combination of neratinib and the radiation energy.
[0095] In one embodiment, the diseased cells may be tumor cells.
The cell proliferation disorder or disease may be breast cancer. In
one embodiment, the tumor cells may be ErbB2+ breast cancer
cells.
[0096] In another embodiment, the radiation energy is UVA or
visible energy which may be applied directly or indirectly.
[0097] In a different embodiment, a psoralen or psoralen derivative
is 8-Methoxypsoralen (8-MOP).
[0098] In yet another embodiment, prior to said applying the
initiation energy, at least one energy modulation agent that
converts the initiation energy to an energy that activates the
psoralen derivative is administered to the subject. The energy
modulation agent is one or more selected from a biocompatible
fluorescing metal nanoparticle, fluorescing dye molecule, gold
nanoparticle, a water soluble quantum dot encapsulated by
polyamidoamine dendrimers, a luciferase, a biocompatible
phosphorescent molecule, a combined electromagnetic energy
harvester molecule, and a lanthanide chelate capable of intense
luminescence.
Discussion
[0099] The molecular basis for the anti-proliferative effects of
photo-activated psoralen in the treatment of benign and neoplastic
skin diseases has historically been attributed to the formation of
interstrand DNA crosslinks that lead to inhibition of transcription
and DNA replication. T cells, which mediate many of the
dermatological indications for PUVA e.g. graft-versus-host disease;
cutaneous T cell lymphoma seem to be particularly sensitive to the
anti-proliferative effects of PUVA and have therefore served as a
frequently used model to study the biological effects of PUVA
therapy [4-6]. In contrast, there has been relatively little
scientific evidence to support the use of PUVA therapy in the
treatment of solid tumors. Here, the inventors have shown for the
first time that PUVA therapy can directly target the catalytic
kinase domain of the ErbB2 receptor tyrosine kinase oncogene. The
interaction of photo-activated 8MOP with regulatory elements within
the ErbB2 catalytic kinase domain may explain the marked inhibition
of ErbB2 signaling in PUVA-treated ErbB2+ breast cancer cells,
including those that have developed resistant to current
FDA-approved ErbB2 targeted therapies.
[0100] The interaction between 8MOP and ErbB2 was demonstrated
using two independent strategies: (i) LC/MS/MS; and (ii) Western
blot analysis. These findings were further supported by the
observation that a DNA non-crosslinking psoralen derivative
maintained its ability to block ErbB2 signaling and induce tumor
cell apoptosis. The ErbB2+ breast cancer cell lines used in these
studies express EGFR, which has also been shown to be a target of
PUVA therapy [9]. However, survival of parental BT474 and SKBR3
cells is not dependent upon EGFR, but instead, dependent upon
signaling via ErbB2-ErbB3 heterodimers [16]. The inventors' data
suggest that the antitumor effects of PUVA therapy in parental
ErbB2+ breast cancer cells were mediated through direct effects on
ErbB2. Moreover, non-malignant HFF cells that express wild-type
EGFR were less sensitive to the apoptotic effects of PUVA,
consistent with the notion that EGFR is not responsible for
induction of apoptosis in PUVA-treated ErbB2+ breast cancer cells
(FIG. 1D).
[0101] Of particular interest is the antitumor activity of PUVA
therapy in ErbB2+ breast cancer models of acquired therapeutic
resistance to lapatinib and other ErbB2 targeted therapies. It is
worth noting that acquired therapeutic resistance to lapatinib does
not appear to be mediated by reactivation of ErbB2 signaling. In
fact, ErbB2 phosphorylation remains inhibited in resistant cells
[16,18]. Importantly, targeted molecular knockdown of ErbB2 does
not reverse lapatinib resistance [18], indicating that survival of
resistant cells is no longer dependent upon ErbB2 alone, at least
not the 185 kDa full-length form of ErbB2 (p185.sup.ErbB2)
expressed at the cell surface. Instead, the viability of lapatinib
resistant ErbB2+ breast cancer cells is dependent upon other
factors. For example, we have shown that resistant cells express a
truncated form of ErbB2, referred to as p85.sup.ErbB2, which can be
generated by alternate initiation of translation [20] and/or
proteolytic processing of p185.sup.ErbB2 [17]. Moreover,
p85.sup.ErbB2 is preferentially expressed in tumor cell nuclei.
This nuclear, truncated form of ErbB2 lacks the extracellular (ECD)
and transmembrane domains, while retaining the full cytoplasmic
domain, including the catalytic kinase domain and tyrosine
autophosphorylation sites. Expression of p85.sup.ErbB2 driven by a
heterologous promoter renders ErbB2+ breast cancer cells that are
normally sensitive to the antitumor effects of lapatinib, resistant
to lapatinib and other ErbB2 targeted therapies. Although the exact
mechanism of p85.sup.ErbB2 action is unknown, it, in contrast to
p185.sup.ErbB2 and p110.sup.ErbB2--a membrane-bound form of ErbB2
that lacks the ECD--does not appear to activate cytoplasmic protein
kinase signaling cascades [17]. The inventors have shown that
tyrosine phosphorylation of p85.sup.ErbB2 is not inhibited by
lapatinib or similar TKIs in class. The inventors have now shown
that PUVA therapy blocks p85.sup.ErbB2 phosphorylation, triggering
apoptosis. One potential explanation is that lapatinib cannot
access the ATP binding groove of p85.sup.ErbB2. In contrast, the
ability of 8MOP to access nuclear targets e.g. DNA, is
well-established. It is therefore possible that 8MOP more readily
accesses, and blocks the catalytic kinase domain of
p85.sup.ErbB2.
[0102] The inventors recently showed that development of lapatinib
resistance can be mediated through a switch in the regulation of
cell survival from ErbB2-ErbB3-PI3K signaling in treatment naive
ErbB2+ breast cancer cells to ErbB3-EGFR-PI3K-PDK1-Akt (T308)
signaling axis in the resistant setting, the latter driven in part
through autocrine production of the ErbB3 ligand heregulin .beta.1
[16]. Although the exact mechanism(s) underlying the antitumor
effects of PUVA is unknown, persistent phosphorylation of Akt T308,
which was seen in models of lapatinib resistance [16], was
inhibited by PUVA. In addition, total EGFR and ErbB3 were reduced
in PUVA-treated resistant cells. These findings are interesting in
light of a recent study showing that several ErbB TKIs can induce
proteolysis of targeted receptor(s) in a manner similar to hsp90
antagonists [21]. Induction of receptor proteolysis by TKIs,
including lapatinib, was shown to be mediated through ATP
competitive binding with cdc37, the latter stimulating binding
between the client protein e.g. ErbB2 and hsp90. It is therefore
possible that PUVA therapy may also trigger degradation of EGFR,
ErbB2, and ErbB3 by blocking access of the cdc37/hsp90 complex to
client proteins ErbB receptors.
[0103] The inventors propose that 8-MOP interacts with the ErbB2
catalytic kinase domain at amino acid residues distinct from
lapatinib. Although structural analysis of a lapatinib-EGFR complex
has been reported, there has been no structural analysis of a
lapatinib-ErbB2 complex. Most of the amino acid residues associated
with the regulation of the ErbB2 catalytic kinase activity are
located in the vicinity of the ATP binding groove within the deep
cleft located between the N- and C-terminal lobes of the ErbB2
receptor [22]. The inventors have found that 8MOP interacts with
peptides located within the DFG motif and activation loop of the
C-lobe, both of which are involved in regulating ErbB2 autokinase
activity [22]. The structural analysis of lapatinib-EGFR crystals
suggests that lapatinib likely interacts with amino acid residues
within the ATP binding groove distinct from those of
photo-activated 8MOP [22]. It is possible therefore that
photo-activated 8-MOP binds to the catalytic kinase domain,
blocking its activity and triggering proteolysis of the receptor in
a manner similar to irreversible ErbB TKIs e.g. neratinib.
[0104] Targeted therapies are increasingly being used in
combination with other targeted and cytotoxic drugs [23]. The
inventors were interested in finding out whether other targeted
therapies might enhance the antitumor activity of PUVA therapy. In
this regard, a recent study found that the combination of a histone
deacetylase (HDAC) inhibitor and PUVA led to enhanced antitumor
activity compared with either treatment alone [24]. When the
inventors examined the effects of adding targeted therapies,
including HDAC and PI3K inhibitors, to PUVA therapy, they found
that neratinib, at sub-lethal doses alone, significantly increased
apoptosis in ErbB2+ breast cancer cells when combined with PUVA. It
is known that neratinib has promiscuous inhibitory activity against
non-ErbB kinases, including MAP kinase family members. It is
possible that the enhanced antitumor effect observed with the
addition of neratinib to PUVA might be directly or indirectly
related to inhibition of a kinase(s) involved in DNA repair of ICL,
thereby sensitizing tumor cells to the DNA damaging effects of
PUVA.
[0105] The data presented here suggests that the antitumor effects
of PUVA can be mediated through DNA-independent mechanisms. It is
possible that inhibition of the ErbB2 signaling axis may sensitize
tumor cells to the DNA damaging effects of PUVA therapy by
inhibiting P3K-Akt regulated DNA damage repair enzymes. In this
context, ErbB2 targeted therapies have previously been shown to
sensitize tumor cells to radiation therapy [25]. Therefore,
therapeutic interventions, including PUVA alone or in combination
with ErbB targeted therapies such as neratinib that can
simultaneously damage DNA and also block ErbB-regulated survival
pathways including those that repair damaged DNA, represent an
attractive therapeutic strategy in treatment naive ErbB2+ tumors
and those that have developed resistance to ErbB2 targeted
therapies through activation of alternate pathways e.g.
ErbB3-EGFR-PDK1-Akt (T308) signaling axis, and express nuclear
truncated ErbB2 receptors that elude the inhibition by existing
ErbB2 targeted therapies.
[0106] As used herein, "pharmaceutically acceptable carrier" is
intended to include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. 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 compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions. Modifications can be made to the compound of the
present invention to affect solubility or clearance of the
compound. These molecules may also be synthesized with D-amino
acids to increase resistance to enzymatic degradation. If
necessary, the activatable pharmaceutical agent can be
co-administered with a solubilizing agent, such as
cyclodextran.
[0107] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, rectal administration, and
direct injection into the affected area, such as direct injection
into a tumor. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerin, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates, and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0108] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, or phosphate buffered saline (PBS). In all
cases, the composition must be sterile and should be fluid to the
extent that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene 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. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, 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, aluminum monostearate and
gelatin.
[0109] 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 that 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, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0110] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0111] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0112] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0113] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0114] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, 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, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0115] It is especially advantageous to formulate oral or
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 subject 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 the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0116] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0117] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only and are not intended to be limiting unless otherwise
specified.
Examples
Cell Culture and Reagents
[0118] ErbB2+(BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human
breast cancer cell lines, and the human foreskin fibroblast (HFF)
cell line were obtained from the American Type Culture Collection
(Manassas, Va.). Lapatinib resistant breast cancer cells (rBT474;
rSKBR3) were generated and maintained in culture as previously
described [16-18]. Cells were maintained in RPMI-1640 supplemented
with 10% fetal bovine serum and L-glutamine from GIBCO (Grand
Island, N.Y.) growing in a humidified atmosphere of 5% CO.sub.2 at
37.degree. C. IRDye 800 conjugated affinity purified anti-rabbit
IgG and anti-mouse IgG were from Rockland (Gilbertsville, Pa.).
Alexa Fluor 680 goat anti-rabbit IgG were purchased from Molecular
Probes (Eugene, Oreg.). Anti-PARP (Poly (ADP-ribose) Polymerase)
monoclonal antibody was from BD PharMingen (San Jose, Calif.).
8-Methoxypsoralen (8MOP) and the 4G10 anti-phosphotyrosine (p-tyr)
antibody were purchased from Sigma-Aldrich (St. Louis, Mo.).
Monoclonal antibodies to c-ErbB2 and EGFR were purchased from Neo
Markers (Union City, Calif.). PARP cleavage product was obtained
from Cell Signaling (Beverly, Mass.). Antibodies to Akt1/2,
phospho-Akt1/2 (S473), phospho-Akt1/2 (T308), phospho-Erk1/2 and
Erk1/2 were purchased from Santa Cruz (Santa Cruz, Calif.).
Lapatinib (GW572016) and neratinib (HKI-272) [19] were purchased
from LC Laboratories (Woburn, Calif.).
[0119] UV Irradiation, Growth/Viability and Apoptosis Assays
[0120] UV irradiation was carried out in 6 or 96 well plate format
in a UV Stratalinker 1800 (Statagene, LA Jolla, Calif.) at the UV
doses indicated in the figures. Cell growth and viability assays
were performed in a 96-well plate format in a final volume of 100
.mu.l/well using the cell proliferation reagent WST-1 from Roche
Diagnostics (Mannheim, Germany), as previously described [16-18].
Details of the apoptosis assay have been previously described
[16-18]. Briefly, cells were treated in 12-well plates with 8MOP,
UV irradiation, or lapatinib at the treatment conditions indicated
in the Figure legends. Cells were harvested with trypsin-EDTA, and
5000 cells in final volume of 50 .mu.l were sampled in 96-well
microplates. Cells were directly stained with annexin V-PE and
nexin 7-AAD in 1.times.Nexin Buffer in a 200 .mu.l final reaction
volume. After incubating at room temperature for 20 min, the
reaction samples were analyzed in the Guava PCA-96-system (Guava
Technology Inc. Hayward, Calif.).
[0121] SDS-PAGE and Western Blot Analysis
[0122] Whole cell extracts were prepared by scraping cells off
petri dishes, washing cell pellets 2.times. in phosphate buffered
saline (PBS), and then re-suspending pellets in two-packed-cell
volumes of RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25%
(w/v) deoxycholate, 1% NP-40, 5 mM sodium orthovanadate, 2 mM
sodium fluoride, and a protease inhibitor cocktail). Protein
concentrations were determined using a modification of the Bradford
method (Bio-Rad Labs, Hercules, Calif.). For Western blot analysis,
equal amounts of proteins (25 to 50 .mu.g) were resolved by either
7.5% or 4-15% gradient SDS polyacrylamide gel electrophoresis under
reducing conditions as previously described [16-18]. Proteins were
transferred to nitrocellulose membranes and probed with primary
antibodies specific for proteins of interest. After extensive
washing, membranes were incubated with a secondary IRDye 800
conjugated anti-rabbit or mouse IgG, or Alexa Fluor 680 anti-rabbit
IgG and proteins were visualized using the LI-COR Odyssey Infrared
Imaging System (LI-COR, Inc., Lincoln, Nebr.).
[0123] Protein Pull-Down and Nano-Flow Liquid Chromatography
Electrospray Ionization Tandem Mass Spectrometry (LC-MS/MS)
Analysis
[0124] BT474 cells were pre-treated with 5 .mu.M biotin-linked
8-MOP for 24 hr before being irradiated with 1J UVA. After UVA
irradiation, cells were harvested and whole cell lysates were
prepared in RIPA buffer. After centrifugation, a 30 .mu.l
suspension of M-280 Streptavidin Dynabeads.RTM. (Invitrogen,
Carlsbad, Calif.) was added to each 130 .mu.l crude lysate sample.
The resulting mixtures were placed on an orbital vortex mixer for
20-30 min. The samples were then magnetized and the supernatants
removed and discarded. A solution of 0.01% (v/v) Tween 20 in PBS
(150 .mu.l) was added to each sample and the resulting mixtures
were placed on orbital vortex mixer for 20-30 min. The samples were
then magnetized and the supernatants were removed and discarded.
Next a solution of 0.1% (v/v) SDS in PBS (150 .mu.l) was added to
each sample and the resulting mixtures were heated to 50.degree. C.
for 15 min. The samples were magnetized and supernatants removed
and discarded, after which 150 .mu.l of 50 mM ammonium bicarbonate
was added to each sample, and the mixtures placed on orbital vortex
mixer for 20-30 min. The samples were again magnetized and the
supernatants removed and discarded. The samples were suspended in
130 .mu.l ammonium bicarbonate (50 mM) prior to mass spectroscopy
analysis. Following a pull-down of biotinylated-drug on immobilized
streptavidin magnetic beads, samples were washed three times with
200 .mu.l 50 mM ammonium bicarbonate, pH 8. Sample volume was
brought to 100 .mu.l 50 mM ammonium bicarbonate (pH 8), and
supplemented with Rapigest surfactant (Waters Corporation, Milford,
Mass.) to a final concentration of 0.1%. Following disulfide
reduction with 5 mM dithiolthreitol at 40.degree. C. for 20 min,
free sulfhydryls were alkylated with 10 mM iodoacetamide at room
temperature for 45 min. Approximately, 500 ng of sequencing grade
modified trypsin (Promega Corporation, Madison, Wis.) was added to
each sample and on-resin digestion was allowed to occur for 18 hr
at 37.degree. C. with orbital shaking. Supernatants were then
collected from each sample after centrifugation at 1000 g for 2 min
and Rapigest surfactant was hydrolyzed by acidification to 0.5%
trifluoracetic acid (final pH 2.5) for 2 hr at 60.degree. C.
Following desalted by C18 Zip-Tip (Millipore) SPE, samples were
brought to dryness by vacuum centrifugation and finally resuspended
in 10 .mu.l 2% acetonitrile, 0.1% formic acid. Peptide mixtures
were subjected to chromatographic separation on a Waters
NanoAcquity UPLC (New Objective, Cambridge, Mass.) equipped with a
1.7 .mu.m BEH130 C.sub.18 75 .mu.m I.D..times.250 mm reversed-phase
column. The mobile phase consisted of (A) 0.1% formic acid in water
and (B) 0.1% formic acid in acetonitrile. Following a 5 .mu.l
injection, peptides were trapped for 5 min on a 5 .mu.m Symmetry
C.sub.18 180 .mu.m I.D..times.20 mm column at 20 .mu.l/min in 99.9%
(A). The analytical column was then switched in-line and a linear
elution gradient of 5% B to 40% B was performed over 90 min at 300
nL/min. The analytical column was connected to a fused silica
PicoTip emitter (New Objective, Cambridge, Mass.) with a 10 .mu.m
tip orifice and coupled to a Waters QToF Premier mass spectrometer
through an electrospray interface. The instrument was operated in a
data-dependent mode of acquisition with the top three most abundant
ions selected for MS/MS using a charge state dependent CID energy
setting with a 60 s dynamic exclusion list employed. Mass spectra
were processed with Mascot Distiller (Matrix Science) and were then
submitted to Mascot searches (Matrix Science) against a
SwissProt_human database appended with reverse entries at 20 ppm
precursor and 0.04 Da product ion mass tolerances with trypsin
protease rules selected. Dynamic mass modifications corresponding
to oxidation on Met residues were allowed. Searched spectra were
imported into Scaffold v2.5 (Proteome Software) and scoring
thresholds were set to yield a protein false discovery rate of 0.2%
(implemented by the PeptideProphet algorithm) based on decoy
database searches.
[0125] Gene Transfection of p85.sup.ErbB2 in Human Breast Cancer
Cells
[0126] The c-terminal fragment (p85.sup.ErbB2) was generated based
on ErbB2 open reading frames and sub-cloned into the pcDNA 3.1 (+)
as previously described [17]. HER2 negative T47D breast cancer
cells were transfected with the p85 expressing vector using the
Lipofectamine.TM. 2000 Reagent from Invitrogen (Carlsbad, Calif.)
as previously described [17]. Stably transfected cells were
selected using G418 (400 .mu.g/rap and the expression level of
p85.sup.ErbB2 was confirmed by western blot analysis as previously
described [17].
[0127] Statistical Analysis
[0128] Data were expressed as means with standard error bars
included. Student's t-test was used to determine statistical
significance between 2 groups. A value of p<0.05 was considered
a statistically significant difference.
Example 1
Inhibition of ErbB2 Signaling Triggers Apoptosis in PUVA-Treated
ErbB2+ Breast Cancer Cells
[0129] The growth and viability of ErbB2+ breast cancer cell lines
was significantly inhibited by PUVA therapy in a dose-dependent
manner (FIGS. 1A and B). The loss of tumor cell viability appeared
to be related to induction of apoptosis (FIGS. 1E and F). In
contrast, PUVA therapy using identical treatment conditions (2.5
and 5 .mu.M 8MOP) had relatively less effect on the growth and
viability of MCF7 cells, a ErbB2 non-overexpressing human breast
cancer cell line and a non-malignant human foreskin fibroblast cell
line (HFF) (FIGS. 1C and D). It was therefore possible that
photo-activated 8MOP might directly modulate ErbB2 activation and
signaling. Seeking to demonstrate the effects of PUVA on ErbB2
signaling, the inventors showed that steady-state protein levels of
the activated, phosphorylated form of ErbB2 were reduced in
PUVA-treated ErbB2+ breast cancer cell lines in a dose-dependent
manner (FIG. 2). In addition, total ErbB2 protein levels were
reduced in response to higher doses of PUVA therapy. In addition,
the activated, phosphorylated forms of Akt and Erk1/2, which are
key downstream mediators of the PI3K and MAPK signaling pathways,
respectively were also inhibited by PUVA (FIG. 2). In contrast,
treatment with the same dose of UV irradiation or 8MOP alone had
relatively little effect on cell viability or ErbB2 signaling
(FIGS. 1 and 2).
Example 2
Psoralen can Directly Interact with the ErbB2 Catalytic Kinase
Domain
[0130] A derivative of psoralen, 7-methylpyridopsoralen (FIG. 3A)
which lacks the DNA binding motif was synthesized, making it unable
to generate ICLs. Next the effects of this compound on ErbB2
signaling and tumor cell viability following UVA irradiation were
determined. Upon photo-activation, 7-methylpyridopsoralen
significantly inhibited the growth and viability of ErbB2+ breast
cancer cells, which correlated with inhibition of phosphorylated
and total ErbB2 protein expression (FIG. 3). These findings
suggested that interruption of ErbB2 signaling in PUVA-treated
tumor cells can be mediated by a mechanism(s) independent of ICL
formation. Next is was sought to determine whether 8MOP can
directly interact with the catalytic kinase domain of ErbB2. In
this regard, BT474 cells were treated with biotinylated-8MOP and a
pull-down experiment was performed (see Materials and Methods).
Biotinylated-8MOP-protein complexes were isolated from BT474 cell
lysate on immobilized streptavidin magnetic beads and subjected to
protein digestion using sequencing grade modified trypsin. Peptides
were then isolated by LC-MS/MS (see Materials and Methods). We
identified three 8MOP bound peptides that corresponded to two sites
located within the catalytic kinase domain (aa 861-868; aa
869-883), and one site in the peptide crossover kinase domain (aa
986-1006) (FIG. 4A). As a second independent approach to
demonstrate the interaction between 8MOP and ErbB2, BT474 cells
were treated with fluorophore-labeled 8MOP (see Materials and
Methods) and UVA irradiation. Cell lysates were separated under
non-denaturing conditions using native gel electrophoresis.
Proteins were transferred to a PVDF membrane, and the
fluorophore-labeled 8MOP detected by an Odyssey scanner (FIG. 4B,
green). The membrane was then blotted with a primary fluoro-labeled
ErbB2 antibody (FIG. 4B, red). The fluoro-conjugated 8MOP was
detected at the same molecular weight as large ErbB2 complexes,
findings that were consistent with the LC-MS/MS data indicating
that 8MOP can directly interact with the ErbB2 receptor.
Example 3
Combination PUVA and Neratinib Treatment Leads to Enhanced Tumor
Cell Killing
[0131] Targeted therapies tend to be more clinically efficacious in
combination with other targeted or cytotoxic drugs. It was
therefore evaluated a variety of targeted agents in combination
with PUVA including PI3K inhibitors, HDAC inhibitors, PARP
inhibitors, and other ErbB TKIs. The most promising combination was
with neratinib (HKI-272), a small molecule, irreversible pan-ErbB
(ErbB1/EGFR; ErbB2; ErbB3; ErbB4) tyrosine kinase inhibitor that is
currently in late phase clinical trials [19]. Treatment of ErbB2+
breast cancer cells with the combination of 8-MOP, UVA irradiation,
and neratinib, each at sub-lethal doses when used alone, resulted
in significantly enhanced inhibition of cell viability (FIG. 5).
The effects of this combination on ErbB2, ErbB3 and downstream
signaling pathways were further analyzed. Consistent with the
inventors' recent findings [16], it was found that neratinib
treatment alone resulted in a marked reduction in total ErbB2 and
ErbB3 protein levels, with consequential loss of p-Akt (S473)
expression. Interestingly, neratinib in combination with UVA
irradiation alone led to further loss of total ErbB2 and ErbB3
protein expression, which was more pronounced in SKBR3 cells (FIG.
5).
Example 4
PUVA Treatment Reverses Lapatinib Resistance in HER2+ Breast Cancer
Cells
[0132] The inventors recently showed that development of acquired
therapeutic resistance to the reversible HER2 and EGFR tyrosine
kinase inhibitor lapatinib in HER2+ breast cancer cells can be
mediated by a number of mechanisms including: (i) a switch in the
regulation of cell survival from HER2-HER3-PI3K signaling in
treatment naive cells to EGFR-HER3-PI3K in resistant cells [16];
and (ii) expression of a truncated ErbB2 form preferentially
expressed in tumor cell nuclei [17]. It was next sought to
determine whether PUVA treatment could reverse lapatinib
resistance. Using models of lapatinib resistance established in our
laboratory [16-18], rBT474 and rSKBR3 cells were treated with PUVA
at the indicated concentrations of 8MOP (FIG. 6A). Resistant cells
were continuously maintained in the presence of 1 .mu.M lapatinib.
As shown, PUVA treatment significantly reduced tumor cell growth
and viability in a dose-dependent manner (FIG. 6A). In contrast to
isotype-matched parental cells (FIG. 5), total EGFR and ErbB3
protein expression was markedly reduced in PUVA-treated rBT474 and
rSKBR3, in addition to reduction in the expression of
phosphorylated forms of EGFR (Y992), ErbB3 (Y1197), and Akt (T308)
(FIG. 6B). The effects of PUVA on Akt T308 are particularly
interesting in light of our recent finding that Akt T308, but not
5473, remained persistently phosphorylated in lapatinib resistant
cells [16]. Importantly, PARP cleavage product was increased in
PUVA-treated lapatinib resistant tumor cells consistent with
induction of apoptosis.
[0133] The inventors have also shown that expression of an 85 kDa
truncated form of ErbB2 (p85.sup.ErbB2) that lacks the
extracellular and transmembrane domains, is preferentially
expressed in the nuclei of tumors that have become resistant to
lapatinib [17]. Moreover, expression of p85.sup.ErbB2 under the
control of a heterologous promoter can render cells resistant to
lapatinib and other ErbB2 targeted drugs in otherwise sensitive
ErbB2+ breast cancer cells [17]. Although p85.sup.ErbB2 is tyrosine
phosphorylation, an indication of its activated state, it is not
inhibited by lapatinib (FIG. 7) [17]. To study the effects of PUVA
on p85.sup.ErbB2, the inventors established a T47D transfected
breast cancer cell that stably expresses phosphorylated
p85.sup.ErbB2 in tumor cell nuclei as previously described [17].
T47D cells, although not HER2 overexpressing, still express
full-length HER2 (FIG. 7). PUVA therapy has an antitumor effect in
T47D cells transfected with empty vector alone that is associated
full-length ErbB2 (p185.sup.ErbB2). However, in p85 expressing T47D
cells, treatment with PUVA, but not lapatinib, markedly inhibited
p85.sup.ErbB2 phosphorylation, triggering tumor cell apoptosis
(FIG. 7).
[0134] Obviously, additional modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
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