U.S. patent application number 12/057097 was filed with the patent office on 2008-10-16 for compositions and methods for protecting cells from toxic exposures.
This patent application is currently assigned to PERSCITUS BIOSCIENCES, LLC. Invention is credited to James P. Thomas.
Application Number | 20080253997 12/057097 |
Document ID | / |
Family ID | 39789046 |
Filed Date | 2008-10-16 |
United States Patent
Application |
20080253997 |
Kind Code |
A1 |
Thomas; James P. |
October 16, 2008 |
COMPOSITIONS AND METHODS FOR PROTECTING CELLS FROM TOXIC
EXPOSURES
Abstract
The present invention provides compositions and methods for
protecting cells and tissues from damage associated with
therapeutic treatments of cancers and other diseases and conditions
where reactive oxygen species are produced. The present invention
also provides compositions useful as research reagents.
Inventors: |
Thomas; James P.; (New
Albany, OH) |
Correspondence
Address: |
Casimir Jones, S.C.
440 Science Drive, Suite 203
Madison
WI
53711
US
|
Assignee: |
PERSCITUS BIOSCIENCES, LLC
Madison
WI
|
Family ID: |
39789046 |
Appl. No.: |
12/057097 |
Filed: |
March 27, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60920176 |
Mar 27, 2007 |
|
|
|
Current U.S.
Class: |
424/85.7 ;
560/147 |
Current CPC
Class: |
C07F 9/1651 20130101;
A61K 41/00 20130101; A61P 39/06 20180101; A61K 31/66 20130101 |
Class at
Publication: |
424/85.7 ;
560/147 |
International
Class: |
A61K 38/21 20060101
A61K038/21; C07C 69/67 20060101 C07C069/67 |
Claims
1. A composition comprising Formula I.
2. The composition of claim 1, wherein R.sub.1 and R.sub.2 groups
are ethyl groups.
3. The composition of claim 1, wherein n is 2.
4. The composition of claim 1, wherein R.sub.1 and R.sub.2 groups
are ethyl groups and wherein n is 2.
5. The composition of claim 1, wherein Formula I further comprises
a monosodium salt of the phosphorothioate group.
6. A composition comprising
2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt.
7. A method for protecting cells from the toxic effects of free
radical generating therapies comprising: a) providing a subject
with a condition being treated with therapies that are toxic to
normal cells and disease cells, b) co-administering to said
subject: a) said toxic therapy and, b) a therapeutic agent, that
through metabolism in said subject, causes accumulation of a
chemoprotectant compound in said normal cells at a higher
concentration than in said disease cells.
8. The method of claim 7, wherein said chemoprotectant compound
comprises glutathione.
9. The method of claim 7, wherein said disease cells comprise
cancer cells.
10. The method of claim 7, wherein said toxic therapy comprises
administration of an anti-cancer chemotherapy.
11. The method of claim 7, wherein said toxic therapy comprises
administration of radiation.
Description
[0001] The present application claims priority to U.S. Provisional
Patent Application Ser. No. 60/920,176, filed Mar. 27, 2007, the
disclosure of which is herein incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention provides compositions and methods for
protecting cells and tissues from damage associated with
therapeutic treatments of cancers and other diseases and conditions
where reactive oxygen species are produced. The present invention
also provides compositions useful as research reagents.
BACKGROUND OF THE INVENTION
[0003] Cancers are a leading cause of death in animals and humans.
The leading cancer therapies today are surgery, radiation and
chemotherapy. In spite of advances in the field of cancer
treatment, each of these known therapies has serious side effects.
For example, surgery disfigures the patient or interferes with
normal bodily functions. Chemotherapy or radiation therapies cause
patients to experience acute debilitating symptoms including
nausea, vomiting, diarrhea, hypersensitivity to light, hair loss,
etc. The side effects of these cytotoxic compounds frequently limit
the frequency and dosage at which they can be administered. The
main reason chemotherapy is so debilitating and the symptoms so
severe is that chemotherapeutic drugs are often unable to
differentiate between normal, healthy cells and the tumor cells
they are designed to target. Therefore, as they target tumor cells
they also target healthy cells thereby causing the toxic side
effects to the subject receiving the chemotherapy. As well,
radiation therapy targets the whole system, not just tumor cells,
so side effects are once again severe for the subject receiving
radiation therapy.
[0004] While chemotherapeutic compounds have been found to be
effective and are in general clinical use as anti-proliferative
agents, there are well recognized drawbacks associated with their
administration. Chemotherapeutic alkylating agents have marked
cytotoxic action and the ability of these drugs to interfere with
normal mitosis and cell division can be lethal. Chemotherapeutic
antimetabolites can lead to anorexia, progressive weight loss,
depression, and coma. Prolonged administration of antimetabolites
can result in serious changes in bone marrow. Both the alkylating
agents and the antimetabolities generally have a depressive effect
on the immunosuppressive system. Prolonged administration of
natural products such as vinca alkyloids can also result in bone
marrow depression. Hydroxy urea and other chemically derived
chemotherapeutic agents can lead to rapid reduction in levels of
adrenocorticosteroids and their metabolites. The administration of
hormonal chemotherapeutic compounds or radioactive isotopes is also
undesirable from the viewpoint of inflicting damage on the
immunosuppressive system and thereby disabling the body's defenses
against common infections. Moreover, it is recently reported that
cognitive function is compromised upon administration of some
chemotherapeutic compounds, in particular the administration of
adriamycin in treating breast cancer. Such cognitive dysfunction is
loosely termed "chemo brain", and is marked by increased oxidative
stress and cellular apoptosis in the brain (Joshi et al., 2007, J.
Neurosci. Res. 85:497-503).
[0005] Glutathione (GSH) represents one of the most prevalent
organic molecules within the cell, with concentrations ranging from
0.1 to 15 mM. Glutathione functions primarily as an antioxidant,
reacting with toxic species as well as serving as a cofactor for a
number of protective enzymes such as glutathione peroxidase and
glutathione transferase. Glutathione is also an important
determinant of the cell's ability to pump toxic substances, such as
chemotherapeutic drug metabolites, out of the cell. The
concentration of glutathione and the extent of glutathione
oxidation are thought to be a key determinant of cells undergoing
programmed cell death (apoptosis) in response to chemotherapy or
radiation therapy.
[0006] Several sulfhydryl containing compounds have been developed
to protect normal tissues from the toxic effects of either
chemotherapy or radiation therapy. For example, glutathione has
been utilized in clinical trials to protect against the toxic
effects of chemotherapy. Cascinu et al. (2002, J. Clin. Once.
20:3478-83) found that co-administration of reduced glutathione
could significantly reduce the neuropathy seen with the
chemotherapeutic drug oxaliplatin. However, the effect of reduced
glutathione is relatively limited in that this compound is rapidly
hydrolyzed when given intravenously. Unfortunately, systemically
administered glutathione protects tumor cells and normal cells
equally and has not been shown to improve the therapeutic index.
Also, elevation in glutathione levels is a common characteristic of
tumor cells resistant to chemotherapy (Moscow and Dixon, 1993,
Cytotech. 12:155-70).
[0007] Sodium 2-mercaptoethane sulphonate (Mesna) is a
thiol-producing compound that is used in clinical oncology to
prevent bladder damage from high doses of chemotherapeutic
alkylating agents (e.g., cyclophosphamide, cisplatin, ifosfamide,
carboplatin, doxorubicin and its derivatives, mitomycin and its
derivatives). Mesna (UROMITEXAN, MESNEX; U.S. Pat. Nos. 5,661,188,
6,696,483 and 6,462,017) is excreted rapidly in the urine which
limits its general utility except for bladder protection.
[0008] Amifostine (ETHYOL, WR-2721; U.S. Pat. Nos. 7,151,094,
6,841,545, 6,753,323, 6,407,278, 6,384,259, 5,994,409) was
developed as a radiation protection agent by the U.S. Walter Reed
Army Institute of Research in the 1950s. Amifostine
(S-2-(3-aminopropylamino)ethylphosphorothioic acid) is a
cytoprotective adjuvant used in cancer chemotherapy involving
DNA-binding chemotherapeutic agents and is used therapeutically to
reduce the incidence of fever and infection induced by DNA-binding
chemotherapeutic agents including alkylating agents (e.g.
cyclophosphamide) and platinum-containing agents (e.g. cisplatin).
It is also used to decrease the cumulative nephrotoxicity
associated with platinum-containing agents and is indicated to
reduce the incidence of dry mouth in patients undergoing
radiotherapy for head and neck cancer. Amifostine is an organic
thiophosphate prodrug that is dephosphorylated in vivo by alkaline
phosphatase (e.g., alkaline phosphatase is capable of hydrolyzing
phosphorothioates in addition to phosphoether moieties in a variety
of compounds) to the active cytoprotective thiol metabolite
(WR-1065). The selective protection of non-malignant tissues is
believed to be due to higher alkaline phosphatase activity, higher
pH, and vascular permeation of normal tissues; dephosphorylation
takes place preferentially in normal blood vessels but to a much
lesser extent in tumor vessels because tumors are more acidic and
the newly formed tumor blood vessels do not significantly express
the enzyme alkaline phosphatase. In randomized Phase III human
trials, amifostine has been shown to reduce toxicity with 1)
chemotherapy and radiation therapy in head and neck cancer (David
et al., 2000, J. Clin. Once. 18:3339-45); 2) radiation therapy in
lung cancer patients (Antonadou et al., 2001, Int. J. Rad. Once.
Biol. Phys. 51:915-22); 3) myelosuppression from carboplatin; and
4) chemotherapy and radiation therapy in rectal cancer. Amifostine
was originally indicated to reduce the cumulative renal toxicity
from cisplatin in non-small cell lung cancer. However, while
nephroprotection was observed, the fact that amifostine could
protect tumors could not be excluded. Therefore, given better
treatment options for non-small cell lung cancer, amifostine's
indication for non-small cell lung cancer was withdrawn in
2005.
[0009] As such, what are needed are novel compositions for use as
broad-spectrum chemoprotectants and radioprotectants. Such novel
compositions would not only serve as adjuvants to chemo and
radiation therapies to protect the subjects normal cells from the
toxicity associated with such therapies, but such novel
compositions would also prove useful as research reagents in the
study of, for example, chemotherapeutics and cellular biology.
SUMMARY OF THE INVENTION
[0010] The present invention provides compositions and methods for
protecting cells and tissues from damage associated with
therapeutic treatments of cancers and other diseases and conditions
where reactive oxygen species are produced. The present invention
also provides compositions useful as research reagents.
[0011] In one embodiment, the compositions of the present invention
are used in conjunction with cytotoxic chemotherapy and/or
radiation therapy in the treatment of subjects, and are broadly
applicable to such treatment regimens. It is contemplated that by
decreasing toxicity to normal cells, the compositions thereby allow
for the escalation (e.g., high dose, prolonged treatment, use of
drugs otherwise considered too toxic, etc.) of chemotherapy or
radiation dosing, resulting in more effective treatments. Likewise,
the compounds find use in conjunction with existing therapeutic
protocols to reduce toxicity and the associated underlying sign,
symptoms, and side effects.
[0012] In some embodiments, the compositions and methods of the
present invention find utility in protecting normal cells from
toxicity due to treatment regimens associated with cellular
toxicity due to, for example, AIDS, anti-fungal therapy,
antibacterial therapy, and intravenous contrast agents. The
compositions and methods can also be used to treat disorders that
are induced by aging and metabolic disorders, including, but not
limited to diabetes.
[0013] The present invention provides compositions and methods for
the treatment of a wide variety of metabolic processes and
disorders wherein free radicals, and therefore cell damage or
apoptosis, can occur. The methods of the present invention are also
suitable for the treatment of disorders relating to basal
metabolism such as heat production of an individual at the lowest
level of cell chemistry in the waking state, or the minimal amount
of cell activity associated with the continuous organic functions
of respiration, circulation and secretion; carbohydrate metabolism
such as the changes that carbohydrates undergo in the tissues,
including oxidation, breakdown, and synthesis; electrolyte
metabolism such as the changes which the various essential
minerals, sodium, potassium, calcium magnesium, etc. undergo in the
fluids and tissues of the body; fat metabolism such as the chemical
changes, oxidation, decomposition, and synthesis, that fats undergo
in the tissues; protein metabolism such as the chemical changes,
decompositions, and synthesis that protein undergoes in the
tissues; and respiratory metabolism such as the exchange of
respiratory gases in the lungs and the oxidation of foodstuffs in
the tissues with the production of carbon dioxide and water.
[0014] In one embodiment, the present invention provides
compositions and methods for protecting tissues and cells from
damage caused by any therapy to a subject that is toxic to normal
cells (e.g., non-diseased cells such as non-cancerous cells), for
example chemotherapy or radiation therapy. In some embodiments, the
present invention inhibits or decreases apoptosis in normal cells
and tissues due to therapies such as, for example, chemotherapy and
radiation therapy.
[0015] In one embodiment, the compositions of the present invention
provide research reagents for the scientific community for use in
experimental methods. In some embodiments, the compositions are
used in in vitro assays. In some embodiments, the compositions are
used in in vivo assays.
[0016] The present invention relates, in part, to compositions and
methods for treating cellular toxicities associated with the
administration to a subject of one or more therapeutic agents,
which comprise administering a therapeutically effective amount of
one or more compositions of the present invention, or
pharmaceutically acceptable salts thereof, to the subject receiving
said one or more therapeutic agents.
[0017] In some embodiments, the therapeutic agent utilized is one
that permits regioselective increase of the concentration of a
natural, non-toxic, protective material in healthy tissue.
Preferably, said compound is not increased, or increased to a
lesser extent, in a cell that is targeted for killing (e.g., a
cancer cell). For example, in some embodiments, the therapeutic
agent provides a regioselective increase in the concentration of
glutathione in healthy tissue. Examples of therapeutic agents that
produce this effect are shown in Formula I, II, and III. The
present invention is not limited to these specific compounds. In
some embodiments, the therapeutic agent is a protected glutathione
molecule that can undergo a selective deprotection process (e.g., a
two-step deprotection process) that locally increases the
concentration of deprotected glutathione in cells of interest
(e.g., healthy tissue). In some embodiments designed to provide
glutathione to cells of interest, the therapeutic agent involves
carboxyl group protection. In some embodiments, one of the carboxyl
groups of glutathione is protected. In some embodiments, both
carboxyl groups of glutathione are protected. In some embodiments,
a phosphorothioate derivative of glutathione is provided, including
mono- and di-ethyl esters thereof. In some embodiments, one or more
methyl or ethyl groups are used to protect one or more carboxyl
groups of a glutathione molecule (see e.g., Formula I, II, and
III). In some embodiments, any protecting group that can be cleaved
(e.g., by a cellular esterase) is employed. Preferably, the product
of the cleavage is minimally toxic or non-toxic. Preferably, the
product is natural glutathione or a functionally equivalent
derivative thereof. In some embodiments, the protecting group is a
polyethylene glycol (PEG). In some embodiments, the protecting
group is any organic moiety that facilitates membrane permeability,
including short peptide or other materials useful for facilitating
drug delivery.
[0018] The present invention is not limited to the use of
glutathione as a protective agent. In some embodiments, the
therapeutic agent is any protective agent that, alone or in
combination with other agents, when modified in vivo in a
regioselective manner, provides a free-radical scavenger in the
desired target cell. For example, the therapeutic agent may
comprise alpha-lipoic acid comprising a phosphate protecting group
or other protecting group (e.g., PEG) protecting the carboxyl
group. A variety of compounds may be employed that can undergo
regioselective deprotection to provide intracellular protective
compounds.
[0019] In some embodiments, the therapeutic agent is provided as
part of a bioconjugate or complex. For example, in some
embodiments, the therapeutic agent is provided in, on, or with a
nanoparticle, liposome, micelle, dendrimer, or other biocompatible
material or biopolymer (e.g., carbohydrate) useful as a drug
carrier.
[0020] In one embodiment, the present invention relates to
compositions and methods for treating cellular toxicities
associated with administration of a chemotherapeutic agent or other
toxic agent wherein a composition comprising Formula I, II, or III,
other compounds described herein, or salts, metabolites, functional
derivatives, functional analogues, esters and pro-drugs thereof,
are administered prior to, with, and/or after administration of the
chemotherapeutic agent, or alternatively, at the first indication
of toxicity caused by the chemotherapeutic agent(s). Toxicity is
caused by, for example, those compounds as listed in Table 1.
[0021] The present invention further relates to methods for
treating cellular toxicities associated with the administration of
therapeutic agents by administering a composition comprising
Formula I, II, or III, other compounds described herein, or salts,
metabolites, functional derivatives, functional analogues, esters
and pro-drugs thereof after clinical appearance of toxicities
following therapeutic treatment. In some embodiments, the invention
relates to methods of treating toxicities associated with the
exposure of a subject to radiation therapy, which comprise
administering to the subject a therapeutically effective amount of
one or more of the compositions as described herein, or a
pharmaceutically acceptable salt thereof, concurrent with, or after
the occurrence of, radiation therapy. In one embodiment, the
present invention relates to compositions and methods for treating
cellular toxicities associated with administration of a radiation
therapy regimen wherein a composition comprising Formula I, II, or
III, other compounds described herein, or salts, metabolites,
functional derivatives, functional analogues, esters and pro-drugs
thereof, are administered prior to, with, and/or after
administration of the radiation therapy, or alternatively, at the
first indication of toxicity caused by the radiation therapy.
[0022] In one embodiment, the present invention provides a
composition comprising Formula I. In some embodiments, Formula I
comprises R.sub.1 and R.sub.2 groups that are each independently
ethyl or methyl groups. In some embodiments, the present invention
provides a composition comprising Formula I wherein n is 2. In some
embodiments, the present invention provides a composition
comprising Formula I wherein R.sub.1 and R.sub.2 groups that are
ethyl groups and n is 2. In some embodiments, Formula I comprises a
monosodium salt of the phosphorothioate group.
[0023] In one embodiment, the present invention provides a
composition comprising
2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt.
[0024] In one embodiment, the present invention provides a method
for protecting cells from the toxic effects of free radical
generating therapies comprising providing a subject with a
conditions being treated with therapies that are toxic to normal
cells and disease cells, and co-administering to said subject a
composition comprising Formula I and a therapy that is toxic to
said normal cells and disease cells.
[0025] In one embodiment, the present invention provides a method
of treating subjects with cancer comprising providing a subject
with cancer and co-administering to said subject a treatment
regimen comprising Formula I and a chemotherapy drug and/or
radiation therapy.
DESCRIPTION OF THE FIGURES
[0026] FIG. 1 depicts a synthesis method of
2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt, an embodiment of the invention,
as described in Example 1.
DEFINITIONS
[0027] As used herein, the term "subject" refers to any animal
(e.g., a mammal), including, but not limited to, humans, non-human
primates, rodents, and the like, which is to be the recipient of a
particular treatment. Typically, the terms "subject" and "patient"
are used interchangeably herein in reference to a human
subject.
[0028] As used herein, the term "non-human animals" refers to all
non-human animals including, but are not limited to, vertebrates
such as rodents, non-human primates, ovines, bovines, ruminants,
lagomorphs, porcines, caprines, equines, canines, felines, aves,
etc.
[0029] As used herein, the term "cell culture" refers to any in
vitro culture of cells. Included within this term are continuous
cell lines (e.g., with an immortal phenotype), primary cell
cultures, transformed cell lines, finite cell lines (e.g.,
non-transformed cells), and any other cell population maintained in
vitro.
[0030] As used herein, the term "in vitro" refers to an artificial
environment and to processes or reactions that occur within an
artificial environment. In vitro environments can consist of, but
are not limited to, test tubes and cell culture. The term "in vivo"
refers to the natural environment (e.g., an animal or a cell) and
to processes or reaction that occur within a natural
environment.
[0031] As used herein, the term "co-administration" refers to the
administration of both a composition of the present invention with
another type of therapy, for example chemotherapy or radiation
therapy. Co-administration can be at the same time in the same
administrative form (e.g., injection, pill, liquid), or
co-administration can be two compositions given at the same time,
but not in the same administrative form.
[0032] As used herein, the term "reactive oxygen species" refers to
highly reactive chemicals, containing oxygen, that react easily
with other molecules, resulting in potentially damaging
modifications. Reactive oxygen species include, for example, oxygen
ions, free radicals and peroxides both inorganic and organic such
as hydrogen peroxide, superoxide, hydroxyl radical, lipid
hydroperoxidase and singlet oxygen. They are generally very small
molecules and are highly reactive due to the presence of unpaired
valence shell electrons.
[0033] As used herein, "toxic effects" refers to damaging
modifications to cells and tissues caused by reactive oxygen
species. For example, a toxic effect of a reactive oxygen species
is a cell that is modified to undergo apoptosis.
[0034] As used herein, "free radical generating therapies" refers
to drugs, chemicals, small molecules, peptides, radiation, and
other such therapies that are applied to subjects, either alone or
in combination, to treat a disorder or disease, wherein such a
therapy results in the generation of free radicals in both
non-diseased and diseased cells and tissues.
DETAILED DESCRIPTION OF THE INVENTION
[0035] Certain illustrative embodiments of the invention are
described below. The present invention is not limited to these
embodiments.
[0036] The compositions of the present invention provide novel
chemoprotectants that, when administered to a subject receiving
chemo or radiation therapy, selectively protects the subject's
cells and tissues, and not tumor tissues, from toxic therapeutic
effects. Once activated, compositions of the present invention
serve, for example, as a direct precursor to glutathione, a key
regulator of apoptosis. The presence of a phosphorothioate moiety,
or other protecting moiety, in the compositions as described herein
requires cleavage by alkaline phosphatase, present in normal cells
but much less so in tumor neovasculature. Elevations of glutathione
in normal tissues render the patient less susceptible to the toxic
effects of chemotherapy and radiation therapy, whereas cancerous
cells within a tumor are not so protected.
[0037] In some embodiments, the compositions as described herein
undergo dephosphorylation (e.g., by alkaline phosphatase) in vitro
under experimental parameters or in vivo in the normal cells and
tissues of a subject. Once dephosphorylated, the composition
comprises an active free sulfhydryl (thiol, --SH) group that
protects against the toxicities associated with chemotherapy and
radiation therapy by acting as a scavenger for reactive oxygen
species created by such therapies (Yuhas, 1977, in: Radiation-Drug
Interactions in Cancer Management, pp. 303-352); Yuhas, 1973, J.
Natl. Cancer Inst. 50:69-78; incorporated by reference herein in
their entireties).
[0038] In one embodiment, the present invention relates to
protection of non-diseased cells and tissues by administering prior
to, during, or after, irradiation and/or chemotherapy to a tumor
tissue, a therapeutically effective amount of a composition as
described herein. In some embodiments, the administration of a
composition of the present invention is directed specifically to
the non-diseased cells and tissues, whereas the administration of
the chemotherapy and/or irradiation is not so discriminating.
[0039] In one embodiment, the compositions of the present invention
include small molecules, or analogs thereof, of the structure as
seen in Formula I:
##STR00001##
wherein: R.sub.1 and R.sub.2 are each, separately, hydrogen,
methyl, or ethyl; and n is an integer from 2 to 10.
[0040] In one embodiment, the present invention provides salts,
solvates and hydrates of the compounds as described herein. An
example of an acceptable salt is found in Formula II:
##STR00002##
wherein: R.sub.1 and R.sub.2 are each, separately, hydrogen,
methyl, or ethyl; and n is an integer from 2 to 10.
[0041] In some embodiments, a further example of a salt composition
suitable for use as a composition in the methods of the present
application is found in Formula III:
##STR00003##
wherein n is an integer from 2 to 10.
[0042] In some embodiments, two or more therapeutic molecules of
interest are provided in a single therapeutic agent as a single
molecule, such that the two or more therapeutic molecules of
interest are generated intracellularly. One or more of the
constituents may also be selected to increase molecule stability,
cell permeability, or other desired properties. For example, in one
embodiment, the compositions of the present invention include small
molecules, or analogs thereof, of the structure as seen in Formula
IV:
##STR00004##
The compound of Formula IV is metabolized to provide both
glutathione and lipoic acid to a cell, each providing protection
against toxic agents or conditions. Such a molecule undergoes, for
example, cleavage of the thiol protecting phosphate by alkaline
phosphatase. It is contemplated that the nonpolar molecule is
readily cell permeable. Esterase cleavage of the conjugate and
liberation of the glutathione molecule and alpha-lipoic acid
provide intracellular protection.
[0043] Therapeutic agents can also be provided as dimers or other
multimers of protective molecules. For example, in some
embodiments, the therapeutic agent comprises a molecule as seen in
Formula V, a protected dimer of glutathione:
##STR00005##
[0044] In some embodiments, compositions of the present invention
are co-administered with chemotherapy and/or anticancer therapy
and/or radiation therapy and another chemoprotectant compound
(e.g., amifostine, mesna). In some embodiments, the administration
of a composition of the present invention is directed specifically
to the non-diseased cells and tissues, whereas the administration
of the chemotherapy and/or irradiation is not so
discriminating.
[0045] For example, Table 1 lists compounds for co-administration
with a composition of the present invention.
TABLE-US-00001 TABLE 1 Aldesleukin Proleukin .RTM. Chiron Corp.,
Emeryville, (des-alanyl-1, serine-125 human CA interleukin-2)
Alemtuzumab Campath .RTM. Millennium and ILEX (IgG1.kappa. anti
CD52 antibody) Partners, LP, Cambridge, MA Alitretinoin Panretin
.RTM. Ligand Pharmaceuticals, (9-cis-retinoic acid) Inc., San Diego
CA Allopurinol Zyloprim .RTM. GlaxoSmithKline,
(1,5-dihydro-4H-pyrazolo[3,4- Research Triangle Park,
d]pyrimidin-4-one monosodium salt) NC Altretamine Hexalen .RTM. US
Bioscience, West (N,N,N',N',N'',N'',-hexamethyl-1,3,5-
Conshohocken, PA triazine-2,4,6-triamine) Amifostine Ethyol .RTM.
US Bioscience (ethanethiol, 2-[(3- aminopropyl)amino]-, dihydrogen
phosphate (ester)) Anastrozole Arimidex .RTM. AstraZeneca
(1,3-Benzenediacetonitrile, a,a,a',a'- Pharmaceuticals, LP,
tetramethyl-5-(1H-1,2,4-triazol-1- Wilmington, DE ylmethyl))
Arsenic trioxide Trisenox .RTM. Cell Therapeutic, Inc., Seattle, WA
Asparaginase Elspar .RTM. Merck & Co., Inc., (L-asparagine
amidohydrolase, type Whitehouse Station, NJ EC-2) BCG Live Tice BCG
.RTM. Organon Teknika, Corp., (lyophilized preparation of an
Durham, NC attenuated strain of Mycobacterium bovis (Bacillus
Calmette-Gukin [BCG], substrain Montreal) bexarotene capsules
Targretin .RTM. Ligand Pharmaceuticals
(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8- pentamethyl-2-napthalenyl)
ethenyl] benzoic acid) bexarotene gel Targretin .RTM. Ligand
Pharmaceuticals Bleomycin Blenoxane .RTM. Bristol-Myers Squibb Co.,
(cytotoxic glycopeptide antibiotics NY, NY produced by Streptomyces
verticillus; bleomycin A.sub.2 and bleomycin B.sub.2) Capecitabine
Xeloda .RTM. Roche (5'-deoxy-5-fluoro-N-
[(pentyloxy)carbonyl]-cytidine) Carboplatin Paraplatin .RTM.
Bristol-Myers Squibb (platinum, diammine [1,1-
cyclobutanedicarboxylato(2-)-0,0']-, (SP-4-2)) Carmustine BCNU,
BiCNU Bristol-Myers Squibb (1,3-bis(2-chloroethyl)-1-nitrosourea)
Carmustine with Polifeprosan 20 Gliadel Wafer Guilford
Pharmaceuticals, Implant Inc., Baltimore, MD Celecoxib Celebrex
.RTM. Searle Pharmaceuticals, (as 4-[5-(4-methylphenyl)-3- England
(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide) Chlorambucil
Leukeran .RTM. GlaxoSmithKline (4-
[bis(2chlorethyl)amino]benzenebutanoic acid) Cisplatin Platinol
.RTM. Bristol-Myers Squibb (PtCl.sub.2H.sub.6N.sub.2) Cladribine
Leustatin .RTM., 2- R. W. Johnson (2-chloro-2'-deoxy-b-D-adenosine)
CdA Pharmaceutical Research Institute, Raritan, NJ Cyclophosphamide
Cytoxan .RTM., Bristol-Myers Squibb (2-[bis(2-chloroethyl)amino]
Neosar .RTM. tetrahydro-2H-13,2-oxazaphosphorine 2-oxide
monohydrate) Cytarabine Cytosar-U .RTM. Pharmacia & Upjohn
(1-b-D-Arabinofuranosylcytosine, Company
C.sub.9H.sub.13N.sub.3O.sub.5) cytarabine liposomal DepoCyt .RTM.
Skye Pharmaceuticals, Inc., San Diego, CA Dacarbazine DTIC-Dome
.RTM. Bayer AG, Leverkusen, (5-(3,3-dimethyl-l-triazeno)-imidazole-
Germany 4-carboxamide (DTIC)) Dactinomycin, actinomycin D Cosmegen
.RTM. Merck (actinomycin produced by Streptomyces parvullus,
C.sub.62H.sub.86N.sub.12O.sub.16) Darbepoetin alfa Aranesp .RTM.
Amgen, Inc., Thousand (recombinant peptide) Oaks, CA daunorubicin
liposomal DanuoXome .RTM. Nexstar Pharmaceuticals,
((8S-cis)-8-acetyl-10-[(3-amino-2,3,6- Inc., Boulder, CO
trideoxy-a-L-lyxo-hexopyranosyl)oxy]-
7,8,9,10-tetrahydro-6,8,11-trihydroxy-
1-methoxy-5,12-naphthacenedione hydrochloride) Daunorubicin HCl,
daunomycin Cerubidine .RTM. Wyeth Ayerst, Madison,
((1S,3S)-3-Acetyl-1,2,3,4,6,11- NJ hexahydro-3,5,12-trihydroxy-10-
methoxy-6,11-dioxo-1-naphthacenyl 3-
amino-2,3,6-trideoxy-(alpha)-L-lyxo- hexopyranoside hydrochloride)
Denileukin diftitox Ontak .RTM. Seragen, Inc., Hopkinton,
(recombinant peptide) MA Dexrazoxane Zinecard .RTM. Pharmacia &
Upjohn ((S)-4,4'-(1-methyl-1,2-ethanediyl)bis- Company
2,6-piperazinedione) Docetaxel Taxotere .RTM. Aventis
Pharmaceuticals, ((2R,3S)-N-carboxy-3-phenylisoserine, Inc.,
Bridgewater, NJ N-tert-butyl ester, 13-ester with 5b-20-
epoxy-12a,4,7b,10b,13a- hexahydroxytax-11-en-9-one 4-acetate
2-benzoate, trihydrate) Doxorubicin HCl Adriamycin .RTM., Pharmacia
& Upjohn (8S,10S)-10-[(3-amino-2,3,6-trideoxy- Rubex .RTM.
Company a-L-lyxo-hexopyranosyl)oxy]-8-
glycolyl-7,8,9,10-tetrahydro-6,8,11- trihydroxy-1-methoxy-5,12-
naphthacenedione hydrochloride) doxorubicin Adriamycin .RTM.
Pharmacia & Upjohn PFS Intravenous Company injection
doxorubicin liposomal Doxil .RTM. Sequus Pharmaceuticals, Inc.,
Menlo park, CA dromostanolone propionate Dromostanolone .RTM. Eli
Lilly & Company, (17b-Hydroxy-2a-methyl-5a-androstan-
Indianapolis, IN 3-one propionate) dromostanolone propionate
Masterone .RTM. Syntex, Corp., Palo Alto, injection CA Elliott's B
Solution Elliott's B Orphan Medical, Inc Solution Epirubicin
Ellence .RTM. Pharmacia & Upjohn
((8S-cis)-10-[(3-amino-2,3,6-trideoxy- Company
a-L-arabino-hexopyranosyl)oxy]-
7,8,9,10-tetrahydro-6,8,11-trihydroxy-
8-(hydroxyacetyl)-1-methoxy-5,12- naphthacenedione hydrochloride)
Epoetin alfa Epogen .RTM. Amgen, Inc (recombinant peptide)
Estramustine Emcyt .RTM. Pharmacia & Upjohn
(estra-1,3,5(10)-triene-3,17- Company diol(17(beta))-, 3-[bis(2-
chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt,
monohydrate, or estradiol 3-[bis(2- chloroethyl)carbamate]
17-(dihydrogen phosphate), disodium salt, monohydrate) Etoposide
phosphate Etopophos .RTM. Bristol-Myers Squibb
(4'-Demethylepipodophyllotoxin 9-[4,6- O-(R)-ethylidene-(beta)-D-
glucopyranoside], 4'-(dihydrogen phosphate)) etoposide, VP-16
Vepesid .RTM. Bristol-Myers Squibb (4'-demethylepipodophyllotoxin
9-[4,6- O-(R)-ethylidene-(beta)-D- glucopyranoside]) Exemestane
Aromasin .RTM. Pharmacia & Upjohn
(6-methylenandrosta-1,4-diene-3,17- Company dione) Filgrastim
Neupogen .RTM. Amgen, Inc (r-metHuG-CSF) floxuridine
(intraarterial) FUDR Roche (2'-deoxy-5-fluorouridine) Fludarabine
Fludara .RTM. Berlex Laboratories, Inc., (fluorinated nucleotide
analog of the Cedar Knolls, NJ antiviral agent vidarabine, 9-b-D-
arabinofuranosyladenine (ara-A)) Fluorouracil, 5-FU Adrucil .RTM.
ICN Pharmaceuticals, Inc., (5-fluoro-2,4(1H,3H)-pyrimidinedione)
Humacao, Puerto Rico Fulvestrant Faslodex .RTM. IPR
Pharmaceuticals, (7-alpha-[9-(4,4,5,5,5-penta Guayama, Puerto Rico
fluoropentylsulphinyl) nonyl]estra-
1,3,5-(10)-triene-3,17-beta-diol) Gemcitabine Gemzar .RTM. Eli
Lilly (2'-deoxy-2',2'-difluorocytidine monohydrochloride
(b-isomer)) Gemtuzumab Ozogamicin Mylotarg .RTM. Wyeth Ayerst
(anti-CD33 hP67.6) Goserelin acetate Zoladex .RTM. AstraZeneca
(acetate salt of [D- Implant Pharmaceuticals
Ser(But).sup.6,Azgly.sup.10]LHRH; pyro-Glu-
His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg- Pro-Azgly-NH2 acetate
[C.sub.59H.sub.84N.sub.18O.sub.14.cndot.(C.sub.2H.sub.4O.sub.2).sub.x
Hydroxyurea Hydrea .RTM. Bristol-Myers Squibb Ibritumomab Tiuxetan
Zevalin .RTM. Biogen IDEC, Inc., (immunoconjugate resulting from a
Cambridge MA thiourea covalent bond between the monoclonal antibody
Ibritumomab and the linker-chelator tiuxetan [N-[2-
bis(carboxymethyl)amino]-3-(p- isothiocyanatophenyl)-propyl]-[N-[2-
bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine) Idarubicin
Idamycin .RTM. Pharmacia & Upjohn (5,12-Naphthacenedione,
9-acetyl-7- Company [(3-amino-2,3,6-trideoxy-(alpha)-L-
lyxo-hexopyranosyl)oxy]-7,8,9,10- tetrahydro-6,9,11-
trihydroxyhydrochloride, (7S-cis)) Ifosfamide IFEX .RTM.
Bristol-Myers Squibb (3-(2-chloroethyl)-2-[(2-
chloroethyl)amino]tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide)
Imatinib Mesilate Gleevec .RTM. Novartis AG, Basel,
(4-[(4-Methyl-1-piperazinyl)methyl]-N- Switzerland
[4-methyl-3-[[4-(3-pyridinyl)-2-
pyrimidinyl]amino]-phenyl]benzamide methanesulfonate) Interferon
alfa-2a Roferon .RTM.-A Hoffmann-La Roche, Inc., (recombinant
peptide) Nutley, NJ Interferon alfa-2b Intron A .RTM. Schering AG,
Berlin, (recombinant peptide) (Lyophilized Germany Betaseron)
Irinotecan HCl Camptosar .RTM. Pharmacia & Upjohn
((4S)-4,11-diethyl-4-hydroxy-9-[(4- Company
piperi-dinopiperidino)carbonyloxy]-1H- pyrano[3',4':6,7]
indolizino[1,2-b] quinoline-3,14(4H,12H) dione hydrochloride
trihydrate) Letrozole Femara .RTM. Novartis
(4,4'-(1H-1,2,4-Triazol-1-ylmethylene) dibenzonitrile) Leucovorin
Wellcovorin .RTM., Immunex, Corp., Seattle, (L-Glutamic acid,
N[4[[(2amino-5- Leucovorin .RTM. WA formyl1,4,5,6,7,8
hexahydro4oxo6- pteridinyl)methyl]amino]benzoyl], calcium salt
(1:1)) Levamisole HCl Ergamisol .RTM. Janssen Research
((-)-(S)-2,3,5,6-tetrahydro-6- Foundation, Titusville, NJ
phenylimidazo [2,1-b] thiazole monohydrochloride
C.sub.11H.sub.12N.sub.2S.cndot.HCl) Lomustine CeeNU .RTM.
Bristol-Myers Squibb (1-(2-chloro-ethyl)-3-cyclohexyl-1-
nitrosourea) Meclorethamine, nitrogen mustard Mustargen .RTM. Merck
(2-chloro-N-(2-chloroethyl)-N- methylethanamine hydrochloride)
Megestrol acetate Megace .RTM. Bristol-Myers Squibb
17.alpha.(acetyloxy)-6-methylpregna-4,6- diene-3,20-dione
Melphalan, L-PAM Alkeran .RTM. GlaxoSmithKline
(4-[bis(2-chloroethyl) amino]-L- phenylalanine) Mercaptopurine,
6-MP Purinethol .RTM. GlaxoSmithKline
(1,7-dihydro-6H-purine-6-thione monohydrate) Mesna Mesnex .RTM.
Asta Medica (sodium 2-mercaptoethane sulfonate) Methotrexate
Methotrexate Lederle Laboratories (N-[4-[[(2,4-diamino-6-
pteridinyl)methyl]methylamino]benzoyl]- L-glutamic acid)
Methoxsalen Uvadex .RTM. Therakos, Inc., Way
(9-methoxy-7H-furo[3,2-g][1]- Exton, Pa
benzopyran-7-one) Mitomycin C Mutamycin .RTM. Bristol-Myers Squibb
mitomycin C Mitozytrex .RTM. SuperGen, Inc., Dublin, CA Mitotane
Lysodren .RTM. Bristol-Myers Squibb
(1,1-dichloro-2-(o-chlorophenyl)-2-(p- chlorophenyl) ethane)
Mitoxantrone Novantrone .RTM. Immunex Corporation
(1,4-dihydroxy-5,8-bis[[2-[(2-
hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione
dihydrochloride) Nandrolone phenpropionate Durabolin .RTM.-50
Organon, Inc., West Orange, NJ Nofetumomab Verluma .RTM. Boehringer
Ingelheim Pharma KG, Germany Oprelvekin Neumega .RTM. Genetics
Institute, Inc., (IL-11) Alexandria, VA Oxaliplatin Eloxatin .RTM.
Sanofi Synthelabo, Inc., (cis-[(1R,2R)-1,2-cyclohexanediamine- NY,
NY N,N'] [oxalato(2-)-O,O'] platinum) Paclitaxel Taxol .RTM.
Bristol-Myers Squibb (5.beta.,
20-Epoxy-1,2a,4,7.beta.,10.beta.,13a- hexahydroxytax-11-en-9-one
4,10- diacetate 2-benzoate 13-ester with (2R,
3S)-N-benzoyl-3-phenylisoserine) Pamidronate Aredia .RTM. Novartis
(phosphonic acid (3-amino-1- hydroxypropylidene) bis-, disodium
salt, pentahydrate, (APD)) Pegademase Adagen .RTM. Enzon
Pharmaceuticals, ((monomethoxypolyethylene glycol (Pegademase Inc.,
Bridgewater, NJ succinimidyl) 11-17-adenosine Bovine) deaminase)
Pegaspargase Oncaspar .RTM. Enzon (monomethoxypolyethylene glycol
succinimidyl L-asparaginase) Pegfilgrastim Neulasta .RTM. Amgen,
Inc (covalent conjugate of recombinant methionyl human G-CSF
(Filgrastim) and monomethoxypolyethylene glycol) Pentostatin Nipent
.RTM. Parke-Davis Pharmaceutical Co., Rockville, MD Pipobroman
Vercyte .RTM. Abbott Laboratories, Abbott Park, IL Plicamycin,
Mithramycin Mithracin .RTM. Pfizer, Inc., NY, NY (antibiotic
produced by Streptomyces plicatus) Porfimer sodium Photofrin .RTM.
QLT Phototherapeutics, Inc., Vancouver, Canada Procarbazine
Matulane .RTM. Sigma Tau (N-isopropyl-.mu.-(2-methylhydrazino)-p-
Pharmaceuticals, Inc., toluamide monohydrochloride) Gaithersburg,
MD Quinacrine Atabrine .RTM. Abbott Labs
(6-chloro-9-(1-methyl-4-diethyl- amine)
butylamino-2-methoxyacridine) Rasburicase Elitek .RTM.
Sanofi-Synthelabo, Inc., (recombinant peptide) Rituximab Rituxan
.RTM. Genentech, Inc., South (recombinant anti-CD20 antibody) San
Francisco, CA Sargramostim Prokine .RTM. Immunex Corp (recombinant
peptide) Streptozocin Zanosar .RTM. Pharmacia & Upjohn
(streptozocin 2-deoxy-2- Company
[[(methylnitrosoamino)carbonyl]amino]- a(and b)-D-glucopyranose and
220 mg citric acid anhydrous) Talc Sclerosol .RTM. Bryan, Corp.,
Woburn, (Mg.sub.3Si.sub.4O.sub.10 (OH).sub.2) MA Tamoxifen Nolvadex
.RTM. AstraZeneca ((Z)2-[4-(1,2-diphenyl-1-butenyl) Pharmaceuticals
phenoxy]-N,N-dimethylethanamine 2-
hydroxy-1,2,3-propanetricarboxylate (1:1)) Temozolomide Temodar
.RTM. Schering (3,4-dihydro-3-methyl-4-
oxoimidazo[5,1-d]-as-tetrazine-8- carboxamide) teniposide, VM-26
Vumon .RTM. Bristol-Myers Squibb (4'-demethylepipodophyllotoxin
9-[4,6- 0-(R)-2-thenylidene-(beta)-D- glucopyranoside])
Testolactone Teslac .RTM. Bristol-Myers Squibb
(13-hydroxy-3-oxo-13,17-secoandrosta- 1,4-dien-17-oic acid
[dgr]-lactone) Thioguanine, 6-TG Thioguanine .RTM. GlaxoSmithKline
(2-amino-1,7-dihydro-6H-purine-6- thione) Thiotepa Thioplex .RTM.
Immunex Corporation (Aziridine, 1,1',1''-
phosphinothioylidynetris-, or Tris (1- aziridinyl) phosphine
sulfide) Topotecan HCl Hycamtin .RTM. GlaxoSmithKline
((S)-10-[(dimethylamino) methyl]-4-
ethyl-4,9-dihydroxy-1H-pyrano[3',4': 6,7] indolizino [1,2-b]
quinoline-3,14- (4H,12H)-dione monohydrochloride) Toremifene
Fareston .RTM. Roberts Pharmaceutical
(2-(p-[(Z)-4-chloro-1,2-diphenyl-1- Corp., Eatontown, NJ
butenyl]-phenoxy)-N,N- dimethylethylamine citrate (1:1))
Tositumomab, I 131 Tositumomab Bexxar .RTM. Corixa Corp., Seattle,
WA (recombinant murine immunotherapeutic monoclonal IgG.sub.2a
lambda anti-CD20 antibody (I 131 is a radioimmunotherapeutic
antibody)) Trastuzumab Herceptin .RTM. Genentech, Inc (recombinant
monoclonal IgG.sub.1 kappa anti-HER2 antibody) Tretinoin, ATRA
Vesanoid .RTM. Roche (all-trans retinoic acid) Uracil Mustard
Uracil Mustard Roberts Labs Capsules Valrubicin, N- Valstar .RTM.
Anthra --> Medeva trifluoroacetyladriamycin-14-valerate
((2S-cis)-2-[1,2,3,4,6,11-hexahydro- 2,5,12-trihydroxy-7
methoxy-6,11- dioxo-[[4 2,3,6-trideoxy-3-
[(trifluoroacetyl)-amino-.alpha.-L-lyxo-
hexopyranosyl]oxyl]-2-naphthacenyl]- 2-oxoethyl pentanoate)
Vinblastine, Leurocristine Velban .RTM. Eli Lilly
(C.sub.46H.sub.56N.sub.4O.sub.10.cndot.H.sub.2SO.sub.4) Vincristine
Oncovin .RTM. Eli Lilly
(C.sub.46H.sub.56N.sub.4O.sub.10.cndot.H.sub.2SO.sub.4) Vinorelbine
Navelbine .RTM. GlaxoSmithKline (3',4'-didehydro-4'-deoxy-C'-
norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate
(1:2)(salt)]) Zoledronate, Zoledronic acid Zometa .RTM. Novartis
((1-Hydroxy-2-imidazol-1-yl- phosphonoethyl) phosphonic acid
monohydrate)
Numerous other examples of chemotherapeutic compounds and
anticancer therapies suitable for co-administration with the
disclosed compositions are known to those skilled in the art.
[0046] In some embodiments, the compositions of the present
invention are especially useful when co-administered with an
anti-cancer drug whose cytotoxicity is due primarily to the
production of reactive oxygen species, for example, doxorubicin,
daunorubicin, mitocyn C, etoposide, cisplatin, arsenic tioxide,
ionizing radiation and photodynamic therapy.
[0047] Anticancer agents further include compounds which have been
identified to have anticancer activity but are not currently
approved by the United States Food and Drug Administration or other
counterpart agencies or are undergoing evaluation for new uses.
Examples include, but are not limited to, 3-AP,
12-O-tetradecanoylphorbol-13-acetate, 17AAG, 852A, ABI-007,
ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG-013736, AGRO100,
alanosine, AMG 706, antineoplastons, AP23573, apaziquone, APC8015,
atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777,
bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib,
bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime,
cetuximab, CGO070, cilengitide, clofarabine, combretastatin A4
phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine,
DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743,
efaproxiral, eflomithine, EKB-569, enzastaurin, erlotinib,
exisulind, fenretinide, flavopiridol, fludarabine, flutamide,
fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein,
glufosfamide, GTI-2040, histrelin, HKI-272, homoharringtonine,
HSPPC-96, iloprost, imiquimod, infliximab, interleukin-12, IPI-504,
irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib,
leuprolide, LMB-9 immunotoxin, lonafarnib, luniliximab,
mafosfamide, MB07133, MDX-010, MLN2704, monoclonal antibody 3F8,
monoclonal antibody J591, motexafin, MS-275, MVA-MUC1-IL2,
nilutamide, nitrocamptothecin, nolatrexed dihydrochloride,
nolvadex, NS-9,06-benzylguanine, oblimersen sodium, ONYX-015,
oregovomab, OSI-774, panitumumab, paraplatin, PD-0325901,
pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS-341,
PSC 833, PXD101, pyrazoloacridine, R115777, RAD001, ranpirnase,
rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4,
rosiglitazone, rubitecan, S-1, S-8184, satraplatin, SB-, 15992,
SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248,
suberoylanilide hydroxamic acid, suramin, talabostat, talampanel,
tariquidar, temsirolimus, TGFa-PE38 immunotoxin, thalidomide,
thymalfasin, tipifarnib, tirapazamine, TLK286, trabectedin,
trimetrexate glucuronate, TroVax, UCN-1, valproic acid, vinflunine,
VNP40101M, volociximab, vorinostat, VX-680, ZD1839, ZD6474,
zileuton, and zosuquidar trihydrochloride.
[0048] For a more detailed description of anticancer agents and
other therapeutic agents, those skilled in the art are referred to
any number of instructive manuals including, but not limited to,
the Physician's Desk Reference, Goodman and Gilman's
"Pharmaceutical Basis of Therapeutics" 10th Edition, Eds. Hardman
et al., 2002 and later editions, and "Biologic Therapy of Cancer,
2nd Edition, Eds. DeVita et al., 1995, JB Lippincott Co. Publ, p.
919 and later editions, incorporated herein by reference in their
entireties.
[0049] In some embodiments, GSH levels in cells, for example both
normal and tumor cells, are reduced prior to the administration of
compounds of Formula I, II, or III. By lowering GSH levels in all
cells, cancer cells become vulnerable to therapies. However,
following treatment with Formula I, II, or III, normal cells are
made substantially more resistant to the toxic effects of the
cancer therapies. Thus, in these embodiments, cancer cells are
supersensitized to therapy, while normal cells are protected. The
present invention is not limited by the nature of the compound or
treatment used to reduce GSH levels.
[0050] In one embodiment, the present invention provides for the
use and administration of 2-amino-4-(S-butylsulfonimidoyl)butanoic
acid (buthionine sulfoximine or BSO) in conjunction with the
compositions of the present invention. In some embodiments,
buthionine sulfoximine inhibits the synthesis of GSH in both
non-tumor and tumor cells by inhibiting .gamma.-glutamulcysteine
synthetase, an essential enzyme for synthesis of GSH, and a
composition of the present invention replenishes GSH in non-tumor
cells. In some embodiments, BSO is administered prior to the
administration of a composition of the present invention. In some
embodiments, BSO is administered in conjunction with a compositions
of the present invention. In some embodiments, the BSO and a
composition of the present invention are administered prior to, at
the same time, or after the administration of chemotherapeutics
and/or radiotherapy to a subject. It is contemplated that as BSO
decreases the amount of GSH in tumor and non-tumor cells, the
addition of a composition of the present invention replenishes GSH
in non-tumor cells but not tumor cells, as such the tumor cells
maintain low or non-existent GSH levels throughout the
administration of chemotherapeutic drugs and/or radiotherapy. The
low or non-existent levels of GSH in tumor cells following
administration of BSO strips them of the protective effects that
GSH offers tumor cells, thereby allowing for more efficient
targeting and eradication of the tumor cells by chemo and radiation
therapies. In some embodiments, the administration of BSO and a
compound of the present invention allows for the administration of
lesser amounts (potentially for longer time periods) of
chemotherapeutic drugs than normal due to the low or non-existent
levels of GSH in tumor cells, and at the same time the non-tumor
cells of a subject are less exposed to the toxic effects of the
therapy.
[0051] In some embodiments, the compositions of the present
invention are useful in preparation as adjuvants to chemo and/or
anticancer therapy and radiation therapy. The methods and
techniques for preparing medicaments comprising a composition of
the present invention are well-known in the art. Exemplary
pharmaceutical formulations and routes of delivery are described
below. One of skill in the art will appreciate that any one or more
of the compounds described herein, including the many specific
embodiments, are prepared by applying standard pharmaceutical
manufacturing procedures. Such medicaments can be delivered to the
subject by using delivery methods that are well-known in the
pharmaceutical arts.
[0052] In some embodiments of the present invention, the
compositions are administered alone, while in some other
embodiments, the compositions are preferably present in a
pharmaceutical formulation comprising at least one active
ingredient/agent, as defined above, together with one or more
pharmaceutically acceptable carriers and optionally other
therapeutic agents. Each carrier should be "acceptable" in the
sense that it is compatible with the other ingredients of the
formulation and not injurious to the subject.
[0053] Formulations include, for example, parenteral administration
(e.g., subcutaneous, intramuscular, intravenous, intradermal) and
site-specific administration. In some embodiments, formulations are
conveniently presented in unit dosage form and are prepared by any
method known in the art of pharmacy. Such methods include the step
of bringing into association the active ingredient with the carrier
that constitutes one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association (e.g., mixing) the active ingredient with liquid
carriers or finely divided solid carriers or both, and then if
necessary shaping the product.
[0054] Formulations suitable for parenteral administration include
aqueous and non-aqueous isotonic sterile injection solutions which
may contain antioxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents, and liposomes
or other microparticulate systems which are designed to target the
compound to blood components or one or more organs. In some
embodiments, the formulations are presented/formulated in unit-dose
or multi-dose sealed containers, for example, ampoules and vials,
and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for
example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared
from sterile powders, granules and tablets of the kind previously
described.
[0055] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention
may include other agents conventional in the art having regard to
the type of formulation in question, for example, those suitable
for oral administration may include such further agents as
sweeteners, thickeners and flavoring agents. It also is intended
that the agents, compositions and methods of this invention be
combined with other suitable compositions and therapies.
[0056] Various delivery systems are known and can be used to
administer compositions of the present invention. Methods of
delivery include, but are not limited to, intra-arterial,
intra-muscular, intravenous, and site specific. For example, in
some embodiments, it may be desirable to administer the
compositions of the invention locally to the area targeted by chemo
and/or anticancer therapies and/or radiation therapy; this may be
achieved by, for example, and not by way of limitation, local
infusion during surgery, injection, or by means of a catheter.
[0057] In some embodiments, in vivo administration of the
compositions as described herein is effected in one dose,
continuously or intermittently throughout the course of treatment.
Methods of determining the most effective means and dosage of
administration are well known to those of skill in the art and vary
with, for example, the composition used for therapy, the target
cell being treated and the subject being treated. Single or
multiple administrations are carried out with the dose level and
pattern being selected by the treating physician. In some
embodiments, the compositions as described herein are delivered to
the subject prior to administration of the chemotherapeutic agent.
In some embodiments, compositions as described herein are delivered
on a daily basis (e.g., at least once, at least twice, at least
three times) and accompany the administration of radiotherapy.
[0058] Suitable dosage formulations and methods of administering
the agents are readily determined by those of skill in the art.
When the compositions described herein are co-administered with
another chemoprotective agent, the effective amount may be less
than when the agent is used alone. Ideally, the agent should be
administered to achieve peak concentrations of the active compound
at the target sites for chemo and radiation therapy. Desirable
blood levels of the agent may be maintained by a continuous
infusion to provide a therapeutic amount of the active ingredient
within the target tissue.
[0059] The present invention also includes methods involving
co-administration of the compositions described herein with one or
more additional active agents. Indeed, it is a further aspect of
this invention to provide methods for enhancing prior art therapies
and/or pharmaceutical compositions by co-administering a compound
of this invention. In co-administration procedures, the agents may
be administered concurrently or sequentially. In one embodiment,
the compounds described herein are administered prior to the other
active agent(s). The pharmaceutical formulations and modes of
administration may be any of those described above. In addition,
the two or more co-administered chemical agents, biological agents
or radiation may each be administered using different modes or
different formulations.
[0060] The agent or agents to be co-administered depends on the
type of condition being treated. For example, when treating cancer,
the additional agent is a chemotherapeutic agent, anticancer agent,
or radiation. The additional agents to be co-administered, such as
anticance can be any of the well-known agents in the art,
including, but not limited to, those that are currently in clinical
use (see Table I for exemplary agents). The determination of
appropriate type and dosage of radiation treatment is also within
the skill in the art or can be determined with relative ease.
[0061] Treatment of the various conditions associated with abnormal
apoptosis is generally limited by the following two major factors:
(1) the development of drug resistance and (2) the toxicity of
known therapeutic agents. In certain cancers, for example,
resistance to chemicals and radiation therapy has been shown to be
associated with inhibition of apoptosis. Some therapeutic agents
have deleterious side effects, including non-specific
lymphotoxicity, renal and bone marrow toxicity.
[0062] The compositions and methods described herein address both
these problems. Drug resistance, where increasing dosages are
required to achieve therapeutic benefit, is overcome by
co-administering the compositions described herein with the known
agent. The compositions described herein protect cells and tissues
from toxic effects of chemotherapeutic drugs and radiation therapy
and, accordingly, less of these agents are needed to achieve a
therapeutic benefit. Conversely, the protection of normal cells and
tissues against the toxic effects of anticancer therapies by
co-administration of the compositions as described herein allows
for higher doses and/or longer treatment regimens when using such
therapies, thereby providing the medical practitioner with the
tools to follow a more aggressive anticancer strategy than was
otherwise deemed possible.
[0063] In some embodiments, the present invention provides methods
for using the compositions as described herein for screening for
the efficacy of such compositions in inhibiting or decreasing
toxicity in cells and tissues when such cells and tissues are
administered cancer, or other, therapies that are toxic to normal
cells. In some embodiments, methods for screening are conducted in
vitro. In other embodiments, these screens are conducted in vivo.
In some embodiments, methods of the present invention are performed
in vivo in non-human animals or human subjects. In some
embodiments, the methods screen for the inhibition or decrease of
apoptosis is cells, in vitro or in vivo, when such cells, non-human
animals, or human subjects are co-administered a cancer, or other,
therapy in combination with compositions of the present invention.
In some embodiments, such methods define efficacy of the
compositions as described herein for use in decreasing or
inhibiting the toxic effects of therapies by comparing results from
a screen with a composition of the present invention to a screen
performed without said composition (e.g., control experiment).
Toxic effects of therapies on cells includes cellular death by
apoptosis as a result of the therapy. A composition of the present
invention that is efficacious in inhibiting or decreasing the toxic
effects of therapies is one that inhibits or decreases cellular
apoptosis in normal, non-diseased cells when toxic therapies are
administered. A skilled artisan will understand methods for
determining cellular apoptosis. These methods include, but are not
limited to, measuring apoptotic indicator enzymes such as caspase
3/7, 8 or 9, TdT-mediated dUTP Nick-End Labeling (TUNEL) assays,
and apoptosis related antibodies (e.g., anti-PARP, anti-caspase 3,
etc.). Detection methods utilized with apoptotic assays include
fluorometric, luminescent, and calorimetric.
[0064] In some embodiments, such in vivo uses are, for example,
performed by taking a subject (e.g., human or non-human animal)
with cancer and co-administering a therapy regimen in conjunction
with a composition of the present invention, and comparing the
outcome of such an administration with a subject that received the
same therapy regimen without co-administration of a composition of
the present invention.
[0065] In some embodiments, such in vitro uses are, for example,
performed in tissue culture dishes with primary or immortalized
tissue culture cells (e.g., HeLa, HEK293, CHO, 3T3, etc.) or tissue
explants. In such in vitro uses, a composition of the present
invention is co-administered with a therapy regimen known to be
toxic to normal cells, the results being compared with results from
tissue culture cells or explants that receive the same therapy
regimen without a composition of the present invention.
EXPERIMENTAL
[0066] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof. In the experimental disclosure which follows, the
following abbreviations apply: equiv (equivalents); M (Molar); N
(Normal); mol (moles); mmol (millimoles); g (grams); L (liters); ml
(milliliters); 0.degree. C. (degrees Centigrade); min. (minutes); %
(percent); psi (pounds per square inch).
Example 1
Preparation of PBS1000
Synthesis of 2-benzyloxycarbonylamino-4-carbamoyl-butyric acid
(1)
[0067] Glutamine (36.5 g, 0.25 mol) was stirred with 1M sodium
bicarbonate (750 ml) and toluene (200 ml). Benzyl chloroformate (50
ml, 59.75 g, 0.35 mol, 1.4 equiv.) was added drop-wise over 20 min.
and the resulting mixture was stirred under nitrogen at room
temperature overnight. Ethyl acetate (400 ml) was added and phases
were separated. The organic phase was extracted with water (50 ml)
and discarded. The aqueous phase was acidified with 6N hydrochloric
acid and extracted with ethyl acetate (2.times.600 ml). The
combined extracts were washed with water (100 ml) and stripped. The
residue was dried in a vacuum oven (50.degree. C.) to produce (1)
(64 g, 91.4%).
Synthesis of 2-benzyloxycarbonylamino-4-carbamoyl-butyric acid
ethyl ester (2)
[0068] A mixture of acid (1) (64 g, 0.228 mol), dimeththylformamide
(210 ml) and sodium bicarbonate (111 g, 1.32 mol, 5.8 equiv.) was
stirred at room temperature for 30 min. Ethyl iodide (34 ml, 66.3
g, 0.425 mol, 1.86 equiv.) was added and stirring was continued
overnight under nitrogen. The reaction mixture was slowly diluted
with water to 1 L and stirred for 40 min. The solid was collected
by filtration, washed well with water and partitioned between ethyl
acetate (8 L) and water (3 L). Phases were separated and the
aqueous phase was extracted with ethyl acetate (2.5 L). The
combined organic extracts were washed with water (1 L), dried over
sodium sulfate, stripped and dried in a vacuum oven (50.degree. C.)
to produce (2) (47 g, 66.7%).
Synthesis of 2-benzyloxycarbonylamino-pentanedioic acid 1-ethyl
ester (3)
[0069] A suspension of the amide (2) (38 g, 0.1233 mol) in
anhydrous acetonitrile (400 ml) was stirred at reflux under
nitrogen and t-butyl nitrite (35 ml, 3.17 equiv.) was added
quickly. The reflux was continued for 2 hrs. After cooling, the
solvent was removed in a rotary evaporator. The residue was taken
in water (250 ml) and ethyl acetate (500 ml) and the biphasic
mixture was stirred well while solid sodium bicarbonate was slowly
added to pH=7.5. Phases were separated and the organic phase was
washed with 10% sodium bicarbonate (200 ml). The combined aqueous
extracts were washed with ethyl acetate (300 ml), made acidic with
6N hydrochloric acid and extracted with ethyl acetate (2.times.300
ml). The combined extracts were washed with water (150 ml), dried
over sodium sulfate, stripped and the residue was dried in a vacuum
oven (50.degree. C.) to produce (3) (24.6 g, 64.7%).
Synthesis of
2-benzyloxycarbonylamino-4-(1-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-bu-
tyric acid ethyl ester (4)
[0070] A suspension of acid (3) (7.6 g, 24.57 mmol) in dry
acetonitrile (80 ml) was stirred under nitrogen at room temperature
and Hobt (4 g, 29.6 mmol, 1.2 equiv.) was added. Stirring was
continued for 10 min., and EDCI (5.1 g, 26.6 mmol, 1.1 equiv.) was
added. The resulting mixture was stirred for 1.5 hrs and serine
ethyl ester free base (3.27 g, 24.57 mmol, 1 equiv.) in
acetonitrile (20 ml) was added. Stirring was continued at room
temperature for 3 hrs. The solvent was removed in a rotary
evaporator, the residue was partitioned between water (100 ml) and
ethyl acetate (200 ml) and phases were separated. The organic phase
was washed successively with water (50 ml), 5% potassium carbonate
(2.times.50 ml) and water (2.times.50 ml), dried over sodium
sulfate and the solvent removed in a rotary evaporator. The residue
was dried in a vacuum oven (50.degree. C.) to produce (4) (12.4 g,
85.5%).
Synthesis of
2-amino-4-(1-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-butyric acid
ethyl ester (5)
[0071] A solution of (4) (11.4 g, 26.86 mmol) in ethanol (225 ml)
containing 1.2 g 20% palladium on activated carbon (50% wet) was
hydrogenated at 30 psi for 3 hrs. The catalyst was removed by
filtration and the solution was washed with ethanol. The solvent
was removed in a rotary evaporator. The residue was dried in a
vacuum oven (50.degree. C.) to produce (5) (5.86 g, 75%).
Synthesis of
3-(2-chloro-1-ethoxycarbonyl-ethylcarbamoyl)-1-ethoxycarbonyl-propyl-ammo-
nium chloride (6)
[0072] A solution of alcohol (5) (0.29 g, 1 mmol) in
dichloromethane (10 ml) was treated with thionyl chloride (1 g) and
stirred at room temperature under nitrogen overnight. The solvent
was removed on a rotary evaporator (bath temperature below
28.degree. C.). Dichloromethane (10 ml) was added and stripped
under the same conditions twice. The solid residue was taken in
water (8 ml) and washed with MTBE (2.times.15 ml). The resulting
aqueous solution contains pure (6) (LCMS) and was used as such in
the next step.
Synthesis of
2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt (7)
[0073] A solution of trisodium thiophosphate (0.4 g) in water (6
ml) was stirred at room temperature under nitrogen and the solution
of (6) prepared above was added all at once. The reaction mixture
was stirred at room temperature under nitrogen overnight. The pH
was carefully adjusted to 8.0 with acetic acid and the resulting
solution was run through a reverse phase column (P18) using water
as the eluent. Fractions were checked by LCMS and those containing
the product were evaporated to dryness (oil pump vacuum, bath
temperature below 25.degree. C.) to produce 47 mg of (7). LCMS
(M=386), .sup.1H NMR and .sup.31P NMR were used to confirm the
final structure (7).
Example 2
Dephosphorylation of
2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt
[0074] Assays were performed to verify the ability of alkaline
phosphatase to dephosphorylate compound (7) to create sulfhydryl
reactive groups. Calf intestinal alkaline phosphatase (CIAP, Sigma)
was diluted in phosphate buffered saline (PBS) to 250 units/ml, and
frozen in tubes containing 100 .mu.l aliquots. The following
solutions were prepared; 2 mM glutathione (GSH), 1.05 mM DTNB
(5-5'-Dithio-bis-(2-nitrobenzoic acid; also known as Ellman's
Reagent) and 5 mM amifostine (AF; 1 mg/ml). Alkaline phosphatase
activity, and the ability of the assay to measure reactive
sulfhydryl groups in solution, were evaluated initially using
amifostine as the control composition. Absorbances were measured at
A.sub.412. An increase in absorbance is indicative of free reactive
sulfhydryl groups present in the reaction. Reaction conditions and
results are found in Table 2; volumes are in .mu.ls, reaction 1 was
incubated for 5 min. at room temperature prior to absorbance
reading, and reactions 2-5 were incubated for 10 min. at room
temperature prior to absorbance readings.
TABLE-US-00002 TABLE 2 REACTION GSH AF CIAP DTNB PBS A412 1 10 100
890 0.35 2 10 890 0.00 3 10 25 865 0.00 4 20 50 100 880 0.03 5 20
50 100 830 0.34
As seen in Table 2, the positive control (reaction 1) and the test
reaction 5 (with amifostine) have similar absorbance readings,
indicating that the reaction conditions are capable of measuring
free sulfhydryl groups after dephosphorylation of a compound with
alkaline phosphatase (reaction 5).
[0075] A second assay was performed to examine the ability of
alkaline phosphatase to dephosphorylate compound (7) to create
sulfhydryl reactive groups. A 12.5 mM solution of Compound 7 was
made (4 mg/ml) and used in the test reactions. Reaction conditions
and results are found in Table 3; volumes are in .mu.ls, reactions
were incubated for 10 min. at 37.degree. C. prior to absorbance
readings, duplicates of the Compound 7 (C7) negative reaction
(without CIAP; reactions 6 & 8) and Compound 7 test reaction
(with CIAP; reactions 7 & 9) were performed.
TABLE-US-00003 TABLE 3 REACTION C7 CIAP DTNB PBS A412 6 10 100 890
0.044 7 10 50 100 840 0.547 8 10 100 890 0.039 9 10 50 100 840
0.526 10 50 100 850 0.015
As seen in Table 3, Compound 7 is dephosphorylated by alkaline
phosphatase to yield free reactive sulfhydryl groups. Such reactive
sulfhydryl groups are capable of capturing free oxygen radicals
created by chemotherapy and/or radiation therapy, thereby
inhibiting or decreasing toxicity of these compounds to normal
cells and tissues. A time course of dephosphorylation was also
performed using Compound 7, following the same reaction conditions
as in Table 3. The time course showed that over a 30 min. period
(A.sub.412 readings taken at 3 min. intervals) the
dephosphorylation of Compound 7 was time dependent, as an increase
in free sulfhydryl groups was seen over time.
Example 3
Intracellular Activity
[0076] The compound
(2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt) was tested for intracellular
properties. In particular, experiments were conducted to determine
the ability of the compound to enter into cells and generate
glutathione. HepG2 were incubated with the compound with or without
added bovine intestinal alkaline phosphatase (Sigma). Cells were
scraped into SSA, vortexed and then spun. GSH in the supernatants
were analyzed utilizing the glutathione reductase method of Tietze
(Tietze F: "ENZYMIC METHOD FOR QUANTITATIVE DETERMINATION OF
NANOGRAM AMOUNTS OF TOTAL AND OXIDIZED GLUTATHIONE APPLICATIONS TO
MAMMALIAN BLOOD AND OTHER TISSUES" Analytical Biochemistry, 27(3):
502-522 (1969)). The compound did not enter cells unless the
phosphate group was first hydrolyzed with alkaline phosphatase.
Cells treated with the compound and alkaline phosphatase had a 3.6
fold increase in their GSH contents. Importantly, this increase in
cellular GSH levels also occurred in the presence of buthionine
sulfoximine (greater than 5 fold increase in cellular GSH),
indicating that the compound was not simply delivering cysteine or
other building blocks for GSH synthesis but rather delivering
gamma-glutamyl cysteine. Cells incubated with compound with or
without alkaline phosphatase did not exhibit any evidence of
toxicity.
[0077] In experiments with mice and hamsters, no overt toxicity was
observed, with testing conducted at doses up to 5
mmoles/animal.
Example 4
Scale-Up Synthesis
[0078] The following example provides a protocol for generating
gram quantities of
2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt.
##STR00006##
L-Glutamine (500 g, 3.42 mol) was stirred with 1M sodium
bicarbonate (10.26 L) and toluene (2.75 L). Benzyl chloroformate
(684 ml, 818 g, 4.8 mol, 1.4 equiv.) was added dropwise over 60
min. and the resulting mixture was stirred under nitrogen at room
temperature overnight. Ethyl acetate (6 L) was added, phases were
separated. The organic phase was extracted with water (1 L) and
discarded. The aqueous phase was made acidic with 6N hydrochloric
acid (11.6 L) and extracted with ethyl acetate (3.times.6 L). The
combined extracts were washed with water (2 L), brine (2 L) and
dried over sodium sulfate. After filtration, the filtrate was
concentrated in vacuo to give a residue which was triturated with
MTBE. The solid was filtered and was dried in a vacuum oven
(45.degree. C.) to yield (823.4 g, 86%) of a solid.
[0079] MS (ESP): 303.0 (M+Na.sup.+) for
C.sub.13H.sub.16N.sub.2O.sub.5
##STR00007##
A mixture (S)-5-amino-2-(benzyloxycarbonylamino)-5-oxopentanoic
acid of (823 g, 2.94 mol), dimethylformamide (3 L) and sodium
bicarbonate (1.481 Kg, 17.6 mol, 6 equiv.) was stirred at room
temperature for 60 min. Ethyl iodide (447 ml, 871 g, 5.6 mol, 1.9
equiv.) was added dropwise over 60 min. and stirring was continued
for 4 days under nitrogen. The reaction mixture was slowly diluted
with water (10 L) and stirred for 60 min. The solid was collected
by filtration, washed with water (8 L) and dried in a convection
oven (50.degree. C.) for 4 days to yield (905 g, 100%) of a
solid.
[0080] MS (ESP): 331.2 (M+Na.sup.+) for
C.sub.15H.sub.20N.sub.2O.sub.5
##STR00008##
A suspension of the (S)-ethyl
5-amino-2-(benzyloxycarbonylamino)-5-oxopentanoate (570 g, 1.85
mol) in anhydrous acetonitrile (6 L) was stirred at reflux under
nitrogen and t-butyl nitrite (650 mL, 3.0 equiv.) was added
quickly. The reflux was continued for 2 hrs. After cooling, the
solvent was removed in a rotary evaporator. The residue was taken
in water (1.5 L) and ethyl acetate (3 L) and the biphasic mixture
was stirred well while solid sodium bicarbonate was slowly added to
pH=7.5. Phases were separated and the organic phase was washed with
10% sodium bicarbonate (6.times.500 ml). The combined aqueous
extracts were washed with ethyl acetate (1 L), made acidic with 6N
hydrochloric acid and extracted with ethyl acetate (4.times.750
ml). The combined extracts were dried over sodium sulfate and
concentrated in vacuo to yield (398 g, 70%) of a solid.
[0081] MS (ESP): 332.0 (M+Na.sup.+) for
C.sub.15H.sub.19NO.sub.6
##STR00009##
A suspension of
(S)-4-(benzyloxycarbonylamino)-5-ethoxy-5-oxopentanoic acid (398 g,
1.29 mol) in dry acetonitrile (4 L) was stirred under nitrogen at
room temperature and HOBt (209 g, 1.54 mol, 1.2 equiv.) was added.
Stirring was continued for 10 min, then EDCI (220 g, 1.42 mol, 1.1
equiv.) was added. The resulting mixture was stirred for 1.5 hrs
when serine ethyl ester free base (171 g, 1.29 mol, 1 equiv.) in
acetonitrile (1 L) was added. Stirring was continued at room
temperature for 16 hrs. The solvent was removed in vacuo and the
residue was partitioned between water (4 L) and ethyl acetate (8 L)
and phases were separated. The organic phase was washed
successively with 5% potassium carbonate (2.times.2 L) and brine
(2.times.2 L), dried over sodium sulfate and the solvent was
removed in vacuo. The residue was triturated with MTBE, filtered
and dried in a vacuum oven (45.degree. C.) to yield (381.4 g, 70%)
as a solid.
[0082] MS (ESP): 447.0 (M+Na.sup.+) for
C.sub.20H.sub.28N.sub.2O.sub.8
##STR00010##
A solution of (S)-ethyl
2-(benzyloxycarbonylamino)-5-((S)-1-ethoxy-3-hydroxy-1-oxopropan-2-ylamin-
o)-5-oxopentanoate (381 g, 0.90 mol) in ethanol (7.5 L) containing
76 g of 10% palladium on activated carbon (50% water wet) was
hydrogenated at 30 psi for 3 hrs. The catalyst was removed by
filtration washing the cake with ethanol (4.times.2 L). The solvent
was removed in vacuo and the residue was triturated with MTBE (2
L), filtered and dried in a vacuum oven (45.degree. C.) to yield
(235.3 g, 91%) of a tan solid.
[0083] MS (ESP): 313.2 (M+Na.sup.+) for
C.sub.12H.sub.22N.sub.2O.sub.6
##STR00011##
A solution of (S)-ethyl
2-amino-5-((S)-1-ethoxy-3-hydroxy-1-oxopropan-2-ylamino)-5-oxopentanoate
(10 g, 35 mmol) in dichloromethane (350 ml) was treated with
thionyl chloride (20 mL) and stirred at room temperature under
nitrogen overnight. The solvent was removed on a rotary evaporator
(bath temperature below 28.degree. C.). Dichloromethane (100 ml)
was added and stripped under the same conditions twice. The solid
residue was triturated with DCM (100 mL), Heptane (100 mL), and
MTBE (100 ml), filtered and dried in a vacuum oven (25.degree. C.)
to yield (10 g, 84%) of an off-white solid.
[0084] MS (ESP): 309.0 (M+H.sup.+) for
C.sub.12H.sub.21ClN.sub.2O.sub.5
##STR00012##
To solution of 40 g of sodium hydroxide in 300 mL of water was
added thiophosphoryl chloride (28.6 g, 0.17 mol) in one portion and
the resulting biphasic solution is quickly heated to reflux. The
reaction mixture is heated at reflux until the thiophosphoryl
chloride layer is no longer observed (approx. 30 min.). The heating
mantle was removed and the reaction mixture cooled to room
temperature. An ice water bath is used to precipitate out the
product and sodium salts (approx. 30 minutes at 0.degree. C.). The
mixture of product and sodium chloride are filtered off, the solids
are collected and dissolved in 150 mL of 45.degree. C. water
(removes sodium chloride). Anhydrous methanol (200 mL) is added to
precipitate the product which is filtered, collected and stirred
under 200 mL of anhydrous methanol for 16 hours to effectively
dehydrate the salt. The solids are again collected by filtration
and dried in a vacuum oven with no heat for 32 hours to yield (17.3
g, 56.5%) of a white solid.
##STR00013##
To a 500 mL round bottom flask was added 250 mL DIUF water. Water
was then degassed with nitrogen over 20 min.
(S)-5-((R)-3-chloro-1-ethoxy-1-oxopropan-2-ylamino)-1-ethoxy-1,5-dioxopen-
tan-2-aminium chloride (5 g, 14.5 mmol) and freshly prepared
trisodiumthiophosphate (2.9 g, 16.0 mmol) were added at once. The
reaction mixture was stirred at room temperature under nitrogen for
3 days. The aqueous mixture was concentrated to a minimal volume in
vacuo keeping the bath temperature below 25.degree. C. The aqueous
residue (50 ml/run) was loaded onto an Analogix 300 g flash C18
column using water as the eluent to yield (6.0 g) of a light yellow
foamy solid that is very hygroscopic.
[0085] MS (ESP): 387.2 (M+H.sup.+) for
C.sub.12H.sub.23N.sub.2O.sub.8PS
[0086] .sup.1H NMR: 1.16 (overlapping triplets, 6H), 2.00-2.21 (m,
3H), 2.31-2.43 (m, 2H), 3.01-3.05 (m, 2H), 3.63-4.20 (m, 7H),
4.41-4.43 (m, 1H); .sup.31P NMR: 17.35 (d)
##STR00014##
In some embodiments, the following steps are used for producing
compound 7 from compound 5. Synthesis of
3-(2-chloro-1-ethoxycarbonyl-ethylcarbamoyl)-1-ethoxycarbonyl-propyl-ammo-
nium chloride (6): A solution of alcohol 5 (10 g, 35 mmol) in
dichloromethane (350 ml) was treated with thionyl chloride (20 mL)
and stirred at room temperature under nitrogen overnight. The
solvent was removed on a rotary evaporator (bath temperature below
28.degree. C.). Dichloromethane (100 ml) was added and stripped
under the same conditions twice. The solid residue was triturated
with DCM (100 mL), Heptane (100 mL), and MTBE (100 ml) to give 10 g
(84% yield) of pure 6 (LCMS) as a off-white solid. Synthesis of
Trisodiumthiophosphate: To a flask was charged 40 g (1.0 mol) of
sodium hydroxide in 300 mL of water. The solution is stirred until
all of the base is dissolved. Thiophosphoryl chloride (28.6 g, 0.17
mol) is added in one portion and the resulting bi-phasic solution
is quickly heated to reflux. The reaction mixture is heated at
reflux until the thiophosphoryl chloride layer is no longer
observed (approx. 30 min). The heating mantle is removed and the
reaction mixture cooled to room temperature. An ice water bath is
used to precipitate out the product and sodium salts (approx. 30
minutes at 0.degree. C.). The mixture of product and sodium
chloride is filtered off, the solids are collected and dissolved in
150 mL of 45.degree. C. water (removes sodium chloride). Anhydrous
methanol (200 mL) is added to precipitate out the
trisodiumphosphoryl chloride. The product is filtered, collected
and stirred under 200 mL of anhydrous methanol for 16 hours to
effectively dehydrate the salt. The solids are again collected by
filtration and dried in a vacuum oven with no heat for 32 hours.
17.3 g of product is obtained in 56.5% yield. Synthesis of
2-amino-4-(1-ethoxycarbonyl-2-phosphonosulfanyl-ethylcarbamoyl)-butyric
acid ethyl ester monosodium salt (7): To a 500 mL round bottom
flask was added 250 mL DIUF water. Water was then degassed by
nitrogen over 20 min. 5 g 6 and 2.9 g fresh made
trisodiumthiophosphate were added at once. The reaction mixture was
stirred at room temperature under nitrogen for 3 days. LC/MS
indicated that the major peak is product. Analogix 300 g flash C18
column was then applied to purify the final product to give 6.0 g
light yellow clear film.
[0087] All publications and patents mentioned in the present
application are herein incorporated by reference. Various
modification and variation of the described methods and
compositions of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention that are obvious to
those skilled in the relevant fields are intended to be within the
scope of the following claims.
* * * * *