U.S. patent application number 14/180281 was filed with the patent office on 2014-06-12 for methods for treatment of thiol-containing compound deficient conditions.
This patent application is currently assigned to NATIONAL JEWISH MEDICAL AND RESEARCH CENTER. The applicant listed for this patent is Brian J. Day, Leonard W. Velsor. Invention is credited to Brian J. Day, Leonard W. Velsor.
Application Number | 20140162986 14/180281 |
Document ID | / |
Family ID | 32179655 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140162986 |
Kind Code |
A1 |
Day; Brian J. ; et
al. |
June 12, 2014 |
Methods for treatment of thiol-containing compound deficient
conditions
Abstract
Methods for therapy of cystic fibrosis and other conditions are
provided. The methods comprise one or more agents capable of
increasing thiol-containing compound transport via a transporter
system (i.e. ABC transporters such as MDR-1 or MRP-2) in cells.
Other embodiments include the use of agents to modulate transport
of thiol-containing compounds within the cell. Therapeutic methods
involve the administration of such agents to a patient afflicted
with cystic fibrosis and/or another condition responsive to
stimulation of thiol-containing compound transport.
Inventors: |
Day; Brian J.; (Englewood,
CO) ; Velsor; Leonard W.; (Greenwood Village,
CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Day; Brian J.
Velsor; Leonard W. |
Englewood
Greenwood Village |
CO
CO |
US
US |
|
|
Assignee: |
NATIONAL JEWISH MEDICAL AND
RESEARCH CENTER
Denver
CO
|
Family ID: |
32179655 |
Appl. No.: |
14/180281 |
Filed: |
February 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10400980 |
Mar 27, 2003 |
|
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14180281 |
|
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Current U.S.
Class: |
514/156 ;
514/159; 514/166 |
Current CPC
Class: |
A61K 31/353 20130101;
A61K 31/35 20130101; A61P 11/00 20180101; A61K 2121/00 20130101;
A61K 31/60 20130101; A61K 31/428 20130101; A61K 31/635 20130101;
A61K 31/37 20130101; A61K 31/47 20130101; A61K 31/606 20130101 |
Class at
Publication: |
514/156 ;
514/166; 514/159 |
International
Class: |
A61K 31/635 20060101
A61K031/635; A61K 31/60 20060101 A61K031/60; A61K 31/606 20060101
A61K031/606 |
Claims
1-54. (canceled)
55. A method for treating inflammatory lung disease, comprising
administering an agent to a subject having inflammatory lung
disease wherein the agent is capable of increasing transport from a
cell of a compound containing thiol-groups, wherein the agent is
selected from sulfasalazine, p-amino salicyclic acid and
5-sulfosalicylic acid and treating the inflammatory lung disease in
the subject.
56. The method of claim 55, wherein the inflammatory lung disease
is an interstitial lung disease.
57. The method of claim 55, wherein the inflammatory lung disease
is cystic fibrosis.
58. The method of claim 55, wherein the transporter system
comprises at least one transporter selected from the group
consisting of an MRP-2 (multi-drug resistance-associated protein)
transporter, an MRP-1 (multi-drug resistance-associated protein)
transporter; an MDR-1 (multi-drug resistance) transporter and
cystic fibrosis transmembrane conductance regulator (CFTR).
59. The method of claim 55, wherein the composition further
comprises a delivery vehicle.
Description
RELATED APPLICATIONS
[0001] The present application claims the benefit under 35 U.S.C.
.sctn.119(e) of provisional U.S. patent application Ser. No.
60/422,802, filed on Oct. 31, 2002.
FIELD
[0002] The present invention relates to methods and compositions
for treatment of cystic fibrosis. More particularly one embodiment
relates to compositions comprising one or more class of agents,
which may be used to activate a thiol-containing compound
transporter (ie. for secretion) in the lung, the intestine, the
pancreas and/or other exocrine glands, and for cystic fibrosis
therapy. Another embodiment relates to an increase in expression of
thiol-containing compound transporter genes or proteins using
molecular biological manupulations and/or application of an
agent(s) to increase the secretion of thiol-containing
compounds.
BACKGROUND
[0003] Cystic fibrosis is a lethal genetic disease afflicting
approximately 30,000 individuals in the United States. Since 1 in
2500 Caucasians is born with cystic fibrosis, it is the most common
lethal, recessively inherited disease in that population. This
inherited disorder impairs epithelial ion transport, particularly
that of chloride. Cystic fibrosis affects the secretory epithelia
of a variety of tissues, altering the transport of water, salt and
other solutes into and out of the blood stream. In particular, the
ability of epithelial cells in the airways, pancreas and other
tissues to transport chloride ions, and accompanying sodium and
water, is severely reduced in cystic fibrosis patients, resulting
in respiratory, pancreatic and intestinal ailments. The principle
clinical manifestation of cystic fibrosis is the resulting
respiratory disease, characterized by airway obstruction due to the
presence of thick mucus that is difficult to clear from airway
surfaces. This thickened airway liquid contributes to recurrent
bacterial infections and progressively impaired respiration. Death
may occur in severe cases because of chronic lung infections,
especially by Pseudomonas aeruginosa, which cause a slow decline in
pulmonary function.
[0004] One current treatment for CF patients focus on controlling
the symptoms of infections through antibiotic therapy and promoting
mucus clearance by use of postural drainage and chest percussion.
However, even with such treatments, frequent hospitalization is
often required as the disease progresses. Thus, long-term therapies
are needed for these patients.
[0005] There are approximately 50 known ATP-binding cassette (ABC)
transporters in humans, and there are currently about 13 genetic
diseases associated with defects in 14 of these transporters. The
most common genetic disease conditions include cystic fibrosis,
Stargardt disease, age-related macular degeneration,
adrenoleukodystrophy, Tangier disease, Dubin-Johnson syndrome and
progressive familial intrahepatic cholestasis. At least 8 members
of this family are involved in the transport of a variety of
amphipathic compounds, including anticancer drugs, and some appear
to contribute to the resistance of cancer cells to chemotherapy.
(Gottesman M M, Ambudkar S V, "Overview: ABC transporters and human
disease." J Bioenerg Biomembr 2001, 33(6):453-8.). ABC transporters
are found in all known organisms, and approximately 1,100 different
transporters belonging to this family have been described in the
literature. The family is defined by homology within the
ATP-binding cassette (ABC) region. Most family members also contain
transmembrane domains involved in recognition of substrates, which
are transported across, into, and out of cell membranes, but some
members utilize ABCs as engines to regulate ion channels.
[0006] Two different integral glycoproteins, the 170 kD
P-glycoprotein (P-gp) and the 190 kD multi-drug resistance protein
(MRP), are involved in the acquisition of multi-drug resistance
phenotypes in cancer cells. Even though they are members of the ABC
superfamily, the primary structures are quite different, only about
15% of the amino acids are identitical. Nevertheless, MRP and P-gp
confer resistance to a similar profile of chemotherapeutic agents
and play a similar role in the acquirement of multi-drug
resistance. Recently, MRP demonstrated the ability to transport the
cysteinyl leukotriene, leukotriene C4 (LTC4) (Ding G Y, Shen T,
Center M S. Multidrug resistance-associated protein (MRP) mediated
transport of daunomycin and LTC4 in isolated plasma membrane
vesicles. Anticancer Res 1999; 19:3243-8.), and other glutathione
conjugates, suggesting that MRP has a function different from P-gp.
MRP is an ATP-dependent glutathione S-conjugate carrier (GS-X pump)
and is present in membranes of many, if not all, cells.
Overexpression of MRP in tumor cells contributes to resistance to
natural product drugs and oxyanions
[0007] In cystic fibrosis, defective chloride transport is
generally due to a mutation in a chloride channel known as the
cystic fibrosis transmembrane conductance regulator (CFTR; see
Riordan et al., Science 245:1066-73, 1989), another member of the
ABC transporter family. CFTR is a linear chloride channel found in
the plasma membrane of certain epithelial cells, where it regulates
the flow of chloride ions in response to phosphorylation by a
cyclic AMP-dependent kinase. Many mutations of CFTR have been
reported, the most common of which is a deletion of phenylalanine
at position 508 (.DELTA.F508-CFTR), which is present in
approximately 70% of patients with cystic fibrosis. A glycine to
aspartate substitution at position 551 (G55 ID-CFTR) occurs in
approximately 1% of cystic fibrosis patients.
[0008] In a healthy lung, glutathione (GSH) is present in high
concentrations in the epithelial lining fluid (ELF) of the lower
respiratory tract, with normal levels in human ELF being more than
200-fold greater than that in plasma. ELF GSH is a major component
of the screening process that protects the pulmonary epithelium
from oxidants released by inflammatory cells as well as inhaled
oxidants. In addition, ELF GSH helps maintain the normal function
of the immune components of the pulmonary epithelial host defense
system. However, in certain conditions, such as idiopathic
pulmonary fibrosis and AIDS patients, a substantial ELF GSH
deficiency exists. Oral administration of GSH does not achieve
significant elevation of GSH level in the lungs and intravenous
administration of GSH is associated with a very short plasma
half-life of the molecule. Thus, a problem exists in supplementing
GSH by conventional means.
[0009] Glutathione (GSH) is a multipurpose mono-thiol compound.
Pure GSH forms a flaky powder that retains a static electrical
charge, due to triboelectric effects, that makes processing
difficult. Glutathione is a strong reducing agent, so that
autooxidation occurs in the presence of oxygen or other oxidizing
agents.
[0010] In synthesizing GSH in the body, cysteine, a thiol amino
acid is required. Since oral administration of glutathione is
ineffective, prodrugs or precursor therapy have been advocated.
Administration of cysteine, or a more bioavailable precursor of
cysteine, N-acetyl cysteine (NAC) was suggested. While cystcine and
NAC are both, themselves, oxygen scavengers, their presence
competes with GSH for resources in certain reducing (GSH recycling)
pathways. Since GSH is a specific substrate for many reducing
pathways, the loading of a host with cysteine or NAC may result in
less efficient utilization or recycling of GSH. Thus, cysteine and
NAC are not ideal GSH prodrugs to solve a deficiency in GSH. Thus,
while GSH may be degraded, transported as amino acids, and
resynthesized in the cell, there may also be circumstances where
GSH is transported into cells without degradation; and in fact the
administration of cysteine or cysteine precursors may interfere
with this process. Thus, loading up on the precurser products is
also a problem.
[0011] A number of disease states have been specifically associated
with reductions in GSH levels. Depressed GSH levels, either locally
in particular organs, or systemically, have been associated with a
number of clinically defined diseases and disease states. These
include HIV/AIDS, diabetes and macular degeneration, all of which
progress because of excessive free radical reactions and
insufficient GSH. Other chronic conditions may also be associated
with GSH deficiency, including heart failure and coronary artery
restenosis post angioplasty.
[0012] Diabetes afflicts 8% of the United States population and
consumes nearly 15% of all United States healthcare costs. HIV/AIDS
has infected nearly 1 million Americans. Current therapies cost in
excess of $20,000 per year per patient, and are rejected by, or
fail in 25% to 40% of all patients. Macular degeneration presently
is considered incurable, and will afflict 15 million Americans by
2002.
[0013] Studies have demonstrated insufficient GSH levels are linked
to these diseases. Newly published data implies that diabetic
complications are the result of hyperglycemic episodes that promote
glycation of cellular enzymes and thereby inactivate GSH synthetic
pathways. The result is GSH deficiency in diabetics, which may
explain the prevalence of cataracts, hypertension, occlusive
atherosclerosis, and susceptibility to infections in these
patients.
[0014] GSH also functions as a detoxicant by forming GSH
S-conjugates with carcinogenic electrophiles, preventing reaction
with DNA, and chelation complexes with heavy metals such as nickel,
lead, cadmium, mercury, vanadium, and manganese. GSH plays a role
in protein folding and deficiencies affect many proteins including
surfactins and dcfensens.
SUMMARY OF THE EMBODIMENTS
[0015] Certain embodiments of the present invention satisfy a need
in the treatment of thiol-containing compound deficient conditions
namely, cystic fibrosis. The embodiments fulfill this need and
further provide other related advantages for other disease
treatments.
[0016] Some of the embodiments provide compositions and methods for
therapy of cystic fibrosis and other conditions. These embodiments
are directed to a method for the modulation of thiol-containing
compound transport in cells. In one embodiment, thiol-containing
compound transport is conferred through over-expression by genetic
manipulation of an ABC transporter. In other embodiments, excretion
of thiol-containing compounds is conferred through increasing the
activity of at least one existing ABC transporter using several
classes of known pharmaceutical agents. Confirmation of transport
is useful to achieve restoration of thiol-containing compounds in
biotechnology applications, and for restoration of thiol-compounds
within cellular compartments, in tissues and whole organs. In other
embodiments, increased secretion of thiol-containing compounds is
used to treat diseases with thiol-containing compound excretion
deficiencies (i.e. cystic fibrosis (CF), idiopathic pulmonary
fibrosis (IPF) and acquired immune deficiency syndrome (AIDS),
pancreatic disease, vascular disease (i.e. vasculitis,
artherosclerosis), intestinal disease (i.e. inflammatory bowel
disease) neurodegenerative disease (i.e. Parkinsons, Alzheimers)
and also male infertility problems).
[0017] Within other aspects of the embodiments, methods for
treating cystic fibrosis in a patient, comprising administering a
compound selected from the group consisting of one of the classes
flavanone, flavone, isoflavone, flavanol, 1,4-naphthoquinone,
3-phenylcoumarin, 2-phenyl-4-quinoline, 1-triflavone, thioflavin,
benzoic acid derivative, indole derivative, naturally occurring
alkaloids, steroids and non-steriod anti-inflammatories (NSAID)
wherein the compound is capable of stimulating thiol-containing
compound transport. Within certain embodiments, the compound may
include but not limited to dexamethasone, rutin, berberine,
biochanin A, indomethacin, propyl gallate, p-aminosalicylatc,
probenacid or sulfasalazine.
[0018] Within further related aspects, method are described for
increasing thiol-compound excretion by airway epithelial cells of a
patient afflicted with cystic fibrosis. One method includes
administering to a mammal one or more compounds selected from
several classes of chemicals for example flavanone, flavone,
isoflavone, isoflavanone 1,4-naphthoquinone, 3-phenylcoumarin,
2-phenyl-4-quinoline, 1-triflavone, thioflavin, benzoic acid
derivative, indole derivative, naturally occurring alkaloids,
steroids and non-steriod anti-inflammatories (NSAID).
[0019] These and other aspects will become apparent upon reference
to the following detailed description and attached drawings.
DEFINITIONS
[0020] The terms "drug resistant" or "drug resistance" as used
herein to describe a property of a cell refer to the ability of the
cell to withstand without cytotoxicity increased concentrations of
a drug as compared to an appropriate control cell. An appropriate
control cell for a cell that has been made drug resistant by
continued exposure to a drug is the parental cell from which the
drug resistant cell was derived. An appropriate control cell for a
cell which has been made drug resistant by expression in the cell
of a protein that confers drug resistance on the cell is the same
cell without the protein expressed. Appropriate control cells for
naturally occurring cells in vivo made drug resistant by continued
exposure to a drug are the same cells at the time of initial
exposure to the drug (parental cell line).
[0021] Homology refers to sequence similarity between sequences and
can be determined by comparing a position in each sequence that may
be aligned for purposes of comparison. When a position in the
compared sequence is occupied by the same nucleotide base or amino
acid, then the molecules are homologous at that position. A degree
of homology between sequences is a function of the number of
matching or homologous positions shared by the sequences.
[0022] The term "sequences having substantial sequence homology"
means those nucleotide and amino acid sequences that have slight or
inconsequential sequence variations from the sequences disclosed
herein (thiol-containing compound transporters) i.e. the homologous
nucleic acids function in substantially the same manner to produce
substantially the same polypeptides as the actual sequences. The
variations may be attributable to local mutations or structural
modifications. It is expected that substitutions or alterations can
be made in various regions of the nucleotide or amino acid sequence
without affecting protein function, particularly if they lie
outside the regions predicted to be of functional significance.
[0023] The term "transformant host cell" is intended to include
prokaryotic and eukaryotic cell that have been transformed or
transfected with a recombinant expression vector. The terms
"transformed with", "transfected with", "transformation" and
"transfection" are intended to encompass introduction of nucleic
acid (e.g. a vector) into a cell by one of many possible
techniques. The recombinant expression vectors can be used to make
a transformant host cell including the recombinant expression
vector. Prokaryotic cells can be transformed with nucleic acid by,
for example, electroporation or calcium-chloride mediated
transformation. Nucleic acid can be introduced into mammalian cells
via conventional techniques such as calcium phosphate or calcium
chloride coprecipitation, DEAE-dextran-mediated transfection,
lipofectin, electroporation or microinjection. Suitable methods for
transforming and transfecting host cells can be found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Laboratory press (1989)), and other laboratory
textbooks).
[0024] As used herein, the terms "engineered" and "recombinant"
cells are intended to refer to a cell into which an exogenous DNA
segment or gene, such as a cDNA or gene has been introduced through
the hand of man. Therefore, engineered cells are distinguishable
from naturally occurring cells that do not contain a recombinantly
introduced exogenous DNA segment or gene. Recombinant cells include
those having an introduced cDNA or genomic gene, and also include
genes positioned adjacent to a heterologous promoter not naturally
associated with the particular introduced gene.
[0025] The term "purified protein or peptide" as used herein, is
intended to refer to a composition, isolatable from other
components, wherein the protein or peptide is purified to any
degree relative to its naturally occurring state (i.e. relative a
cell extract). A purified protein or peptide therefore also refers
to a protein or peptide, free from the environment in which it may
naturally occur.
[0026] Generally, "purified" will refer to a protein or peptide
composition which has been subjected to fractionation to remove
various other components, and which composition substantially
retains its expressed biological activity. Where the term
"substantially purified" is used, this will refer to a composition
in which the protein or peptide forms the major component of the
composition, such as constituting about 25% or more of the proteins
in the composition.
[0027] The term "subject" is intended to include living organisms
in which an immune response can be elicited, e.g., mammals.
Examples of subjects include humans, dogs, cats, rats, mice and
transgenic species thereof.
[0028] The phrases "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce an adverse, allergic or other untoward reaction when
administered to an animal, or a human, as appropriate.
[0029] The term "unit dose" refers to physically discrete units
suitable for use in a subject, each unit containing a
pre-determined-quantity of the therapeutic composition calculated
to produce the desired responses, discussed above, in association
with its administration, i.e., the appropriate route and treatment
regimen. The quantity to be administered, both according to number
of treatments and unit dose, depends on the subject to be treated,
the state of the subject and the protection desired. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject.
[0030] "Liposome" is a generic term encompassing a variety of
single and multilamellar lipid vehicles formed by the generation of
enclosed lipid bilayers. Phospholipids are used for preparing the
liposomes accordingly and can carry a net positive charge, a net
negative charge or are neutral. Dicetyl phosphate can be employed
to confer a negative charge on the liposomes, and stearylamine can
be used to confer a positive charge on the liposomes.
[0031] The term "flavones", as used herein refers to a compound
based on the core structure of flavone. Non-limiting examples of
flavones encompassed by this invention are apiin, myricetin,
quercetin, luteolin, rutin, kampferol, and apigenin.
[0032] An "isoflavone" is an isomer of a flavone (i.e., the phenyl
moiety at position 2 is moved to position 3), and having the core
structure shown below. Non-limiting examples of isoflavones
encompassed by this invention are genistein, daidzein, biochanin A,
baptigenin and formononetin.
[0033] A "flavanone" is an isomer of flavone (the C ring is not
aromatic), and have the core structure shown below. Non-limiting
examples of flavanones encompassed by this invention are taxifolin,
naringenin, naringin, eriodictyol, and fustin.
[0034] A "flavanol" is an isomer of flavanone (the C ring is not
aromatic and lacks an oxo group) and having the core structure
shown below: An example is catechin.
##STR00001##
[0035] The term "benzoic acid derivatives" as used herein refers to
a compounds based on the core structure of benzoic acid. Examples
and structures (one or more carboxylic acid group(s) can be
substituted at any of the 6 carbons of the benzene ring) are shown
below:
Benzoic Acid Derivatives
##STR00002##
[0037] Acetylsalicylsaliclic acid (2-(aceyl-oxy)benzoic acid
2-carboxyphenyl ester)
[0038] Ambucaine (4-amino-2-butoxybenzoic acid 2-diethylaminoethyl
ester)
[0039] p-Aminosalicylic acid (4-amino-2-hydroxybenzoic acid)
[0040] p-Aminosalicylic acid hydrazide (4-amino-2-hydroxybenzoic
acid hydrazide)
[0041] p-Aminosulfobenzoic acid (4-amino-2-sulfobenzoic acid)
[0042] Anacardic acid
[0043] p-Anisic acid (4-methoxybenzoic acid)
[0044] o-(p-Anisoyl)benzoic acid (2-(-4-methoxybenzoyl)benzoic
acid)
[0045] Aspirin (2-(acetyloxy)benzoic acid)
[0046] Avobenzonc
(1-[4-(1,1-dimethylethyl)phenyl]-3-(4-methoxyphenyl)-11,3-propanedione
[0047] Benzoic acid
[0048] Benzonatate (4-(butylamino)benzoic acid)
[0049] Benzoylpas (4-(benzoylamino)-2-hydroxybenzoic acid)
[0050] Benzyl salicylate (2-hydroxybenzoic acid
phenylmethylester)
[0051] Betoxycaine (3-amino-4-butoxybenzoic acid
2-[2-(dimethylamno)ethoxy]ethyl ester)
[0052] m-,o-, p-Chlorobenzoic acid
[0053] m-,o-, p-Cresotic acid
[0054] Cuelurc (4-[4-(acetyloxy)phenyl]-2-butanone)
[0055] Cumic acid (4-(1-methylethyl)benzoic acid)
[0056] Difunisal
(2',4'-difluoro-4-hydroxy-[1',1'-biphenyl]-3-carboxylic acid)
[0057] Ethylparaben (4-hydroxybenzoic acid ethyl ester)
[0058] Gallic acid (3,4,5-trihydroxybenzoic acid)
[0059] m-,o-,p-Hydroxybenzoic acid
[0060] Mesalamine (5-amino-2-hydroxybenzoic acid)
[0061] Methylparaben (4-hydroxybenzoic acid methylester)
[0062] Methyl salicylate (2-hydroxybenzoic acid methyl esther)
[0063] o-Orsellinic acid (2,4-dihydroxy-6-methylbenzoic acid)
[0064] Propyl gallate (3,4,5-trihydroxy-benzoic acid propyl
ester)
[0065] Propylparaben (4-hydroxybenzoic acid propyl ester)
[0066] Salicylic acid (2-hydroxybenzoic acid)
[0067] Salicylsulfuric acid (2-(sulfooxy)benzoic acid)
[0068] Salsalate (2-hydroxybenzoic acid carboxyphenyl ester)
[0069] Sulfosalicylic acid (5-hydroxy-5-sulfo-benzoic acid)
[0070] Thiosalicylic acid (2-mercaptobenzoic acid)
[0071] Vanillic acid (4-hydroxy-3-methoxybenzoic acid)
[0072] The term "indole derivatives" as used herein refers to a
compounds based on the core structure of indole. Examples and
structures are shown below:
[0073] Indole Derivatives
##STR00003## [0074] Adrenolutin (1-methyl-1H-indole-3-5,6-triol)
[0075] Aminochromes (2,3-dihydroindole-5,6-quinone) [0076]
5-Hydroxytryptophan [0077] Hypaphorinc
(1-trimethyl-ammonio-3-(3-indolyl)propionate) [0078] Indalpine
(3-[2-(4-piperidinyl)ethyl]-1H-indole) [0079] Indapamide
(3-(aminosulfonyl)-4-chloro-N-(2,3-dihydro-2-methyl-1H-indol-1-yl)benzami-
d [0080] Indican (indol-3-yl sulfate) [0081] Indican
(3-(.beta.-glucosido)indole) [0082] Indigo
(2-(1,3-dihydro-3-oxo-2H-indol-2-ylidene)-1,2-dihydro-3H-indol-3-one))
[0083] Indigo Carmine
(2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-in-
dole-5 sulfonic acid) [0084] Indo-1
(2-[4-[Bis(carboxymethyl)-amino]-3-[2-[2-[bis(caroxymethyl)amino]-5-methy-
lphenoxyl]-ethoxy]phenyl]-1H-indole-6-carboxylic acid) [0085]
Indobufen
(4-(1,3-dihydro-1-oxo-2H-isoindol-2-yl)-.alpha.-ethylbeneneacetic
acid [0086] Indole (2,3-benzopyrrole) [0087] Indoleacetic acid
(1H-indole-3-acetic acid) [0088] Indolebutyric acid
(1H-indole-3-butanoic acid) [0089] Indolmycin
((5S)-5-[(1R)-1-(1H-indol-3-yl)ethyl]-2-(methylamino)-4(5H)-oxazolone)
[0090] Indomethacin
(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid)
[0091] Indoprofen
(4-(1,3-dihydro-1-oxo-2H-isoindol-2-yl)-.alpha.-methylbenzeneacetic
acid) [0092] Indoramin
(N-[1-(2-[1H-indol-3-yl)ethyl]-4-piperidinyl]benzamide) [0093]
Isatin (indole-2,3-dione) [0094] Psilocin
(3-[2-(dimethylamino)ethyl]-1H-indol-4-ol) [0095] Psilocybin
(3-[2-(dimethylamino)ethyl]-1H-indol-4-ol) [0096] Serotonin [0097]
Skatole (3-methyl-1H-indole)
[0098] Other compounds that pertain to the embodiments include
naphthoquinones, coumarins quinoline thioflavones, thioflavins,
glucocorticiods, steroid, naturally occurring alkaloids also MMP
(matrix metalloproteinase) inhibitors and xenobiotics (i.e.
pesticides) are included.
[0099] Glucocorticoids are adrenocortical steroids, both naturally
occurring and synthetic, which are readily absorbed from the
gastrointestinal tract. Dexamethasone, a synthetic adrenocortical
steroid and is stable in air. The molecular weight is 392.47. It is
designated chemically as
9-fluoro-11b,17,21-trihydroxy-16a-methylpregna-1,4-diene-3,20-dione.
The empirical formula is C.sub.22H.sub.29FO.sub.5.
[0100] Many of the above named chemicals are naturally occurring,
but synthetic compounds are also encompassed. The chemical may be
modified to include any of a variety of functional groups, such as
hydroxyl and/or ether groups. Preferred chemicals such as flavones
include one or more hydroxyl groups, such as the trihydroxyflavone
apigenin, the tetrahydroxyflavone kaempferol and the
pentahydroxyflavone quercetin. Preferred isoflavones include one or
more hydroxyl groups, such as trihydroxyisoflavone genistein and
methoxy containing biochanin A.
BRIEF DESCRIPTION OF THE DRAWINGS
[0101] FIG. 1 illustrates cellular synthesis, metabolism and
transport of glutathione (GSH) into the mitochondria.
[0102] FIG. 2 represents a schematic example of possible
biochemical consequences of oxidative stress.
[0103] FIG. 3 represents a schematic of the progression of Cystic
Fibrosis (CF) and lung disease.
[0104] FIG. 4 represents a schematic of detoxification of peroxides
by the glutathione redox cycle.
[0105] FIG. 5 represents pulmonary ELF concentrations of GSH in
Cystic Fibrosis Transmembrane Regulator Protein Knockout (CFTR KO)
mice compared to a control.
[0106] FIG. 6 represents the levels of Glutathione Reductase
activity in control versus CFTR KO mice.
[0107] FIG. 7 represents the levels of Glutathione Peroxidase
activity in control versus CFTR KO mice.
[0108] FIG. 8 represents oxidation of DNA in the lungs of control
versus CFTR KO mice.
[0109] FIG. 9 represents the levels of mitochondrial Aconitase
activity in control versus CFTR KO mice.
[0110] FIG. 10 represents the concentration of lipid peroxidation
in the lungs of control versus CFTR-KO mice.
[0111] FIG. 11 represents lung and intestinal mitochondrial GSH in
control versus CFTR KO mice.
[0112] FIG. 12 represents a schematic of cystic fibrosis and the
effects of GSH in the progression to lung failure.
[0113] FIG. 13 illustrates a schematic of a hepatocyte and several
transporters for detoxification used in multi-drug resistance.
[0114] FIG. 14 represents a schematic of cellular synthesis,
metabolism and transport of GSH.
[0115] FIG. 15. represents Pseudomonas killing by an eight-hour
exposure to mouse bronchoalveolar lavage fluid (BALF)
[0116] FIG. 16. represents the effects of Rutin and Dexamethasone
on the extracellular concentration of GSH
[0117] FIG. 17 represents the levels of lung ELF GSH in control
versus Dexamethasone treated mice.
[0118] FIG. 18 represents the effect of Pseudomonas endobronchial
infection on lung ELF GSH levels and induction of lung MRP2
transporter and CFTR expression.
[0119] Table 1. represents a list of amino acids and codon
usage.
[0120] Table 2 illustrates proposed apical lung GSH and other
thiol-containing compound transporters.
[0121] Table 3. represents chemical compounds and their effects on
extracellular GSH transport.
DETAILED DESCRIPTION
[0122] In the following description, several specific details are
presented such as examples of specific methods, components, and
processes in order to provide a thorough understanding of various
embodiments. It will be obvious to one skilled in the art that
these specific details need not be employed to practice the various
embodiments. In other cases, some well-known components or methods
will not be described in detail in order to alleviate unnecessary
obscuring of various embodiments presented forthwith.
[0123] Compositions and methods of use to treat thiol-containing
compound transport deficiencies are described.
[0124] Cystic Fibrosis (CF) is a devastating genetic disorder that
results in chronic infection of the lung with a deteriorating cycle
of inflammation and injury that ultimately destroys the lung. CF is
caused by mutations in the protein called CFTR, cystic fibrosis
transmembrane conductance regulator, an ABC-transporter-like
protein found in the plasma membrane of animal cells (Rommens, J.
M. et al. (1989) Science 245:1059-1065; Riordan, J. R. et al.
(1989) Science 245:1066-1073; Kerem, B-S. et al. (1989) Science
245:1073-1080). The CFTR gene is localized within a putative ATP
binding/ATP hydrolysis domain. The deletion of phenylalanine at
position 508 (.DELTA.F508-CFTR) represents approximately 70% of
patients with cystic fibrosis. CFTR is an integral membrane protein
primarily expressed in the epithelia of the lung, pancreas, sweat
glands, and vas deferens. Recently, other than transport of
chloride ions, this transporter also carries glutathione to the
cell exterior. In the lung the epithelial cells maintain the
epithelial lining fluid (ELF) that coats the airways and is a
critical component of the lung host defense that helps the
mucociliary clearance pathway. This pathway is critical in
providing a sterile environment in the lung and is severely
compromised in CF patients.
[0125] Recent studies suggest that the CFTR protein modulates ELF
GSH and when defective creates an imbalance of glutathione-mediated
processes in the lung. CF patients bearing this deltaF508 mutation
frequently experience chronic lung infections, particularly by
Pseudomonas aeruginosa, and have a limited life span. Attempts to
remedy the mutated CFTR protein have been unsuccessful. The
deltaF508 mutation destroys the proteins ability to function as a
transporter. The embodiments of this invention identify therapies
that address alternate treatments for CF affected individuals and
ELF replenishment of thiol-containing compound levels including the
stimulation of other unaffected transporters, as well as therapies
for other thiol-compound excretion deficient afflicted
patients.
[0126] Other complications may arise in CF patients such as they
suffer from diminished pancreatic function that leads to inadequate
breakdown and absorption of fat-soluble nutrients. Poor absorption
may result in deficiencies of the fat-soluble antioxidant vitamins
for example .alpha.-tocopherol (vitamin E) and .beta.-carotene
(precurser of vit. A), as well as other components of the oxidant
scavenging system such as ferritin and selenium.
[0127] Other than CF patients, other thiol-containing compound
deficient conditions exist. An immune-compromised or intensive care
unit patient has below normal cellular levels of GSH. It is
believed that a patient who has decreased GSH levels is more
susceptible to many disease states. It is therefore important to
ensure that extracellular and intracellular GSH levels are
maintained at near normal levels, or increased to meet those
levels.
[0128] A number of such patients having reduced glutathione levels
also have impaired or compromised gut functions. Examples of such
patients include those suffering from: AIDS; Crohn's disease;
chronic inflammatory bowel disease (IBD); short bowel syndrome; and
inflammatory bowel reaction to radiation therapy. Providing an
intact protein, such as casein, does not provide a sufficiently
bioavailable source of GSH to the patient since the gut function of
these patients is compromised. Thus, supplying adequate amounts of
thiol-containing compounds to these distressed areas is critical in
patient recovery.
[0129] The status of reduced glutathione
(L-.gamma.-glutamyl-L-cysteinyl-glycine, GSH), in ELF of adults
with CF has been evaluated. In normal individuals, respiratory ELF
has high levels of GSH, typically 200-fold greater than plasma
(Cantin, A. M. et al. (1987) J. Appl. Physiol. 63:152-157). There
is a chronic influx of oxidants on the respiratory epithelium, and
with the knowledge that oxidants released from inflammatory cells
can derange the respiratory epithelial structure and function and
interfere with host defense GSH can scavenge all major oxidants
produced by inflammatory cells (Meister, A. (1988) J. Biol. Chem.
263:17205-17208; Buhl, R. et al. (1990) Proc. Natl. Acad. Sci. USA.
87:4063-4067; Heffner, J. E., and J. E. Repine (1989) Am. Rev.
Respir. Dis. 140:531-554), and its function as an antioxidant on
the respiratory epithelial surface is enhanced by the presence of
glutathione peroxidase and glutathione reductase in respiratory ELF
(Meister, A. (1988) J. Biol. Chem. 263:17205-17208; Cantin, A. M.
et al. (1990) J. Clin. Invest. 86:962-971; Davis, W. B., and E. R.
Pacht (1991) In The Lung: Scientific Foundations. R. G. Crystal and
J. B. West, editors. Raven Press, New York. 1821-1828). GSH is
believed to be the primary intracellular antioxidant for higher
organisms. It is a mono-thiol compound. When oxidized, it forms a
dimer (GSSG), which is likely recycled into cells having
glutathione reductase (Tanuguchi, N., et al. (1989., Glutathione
Centennial, Academic Press, New York).
[0130] GSH is synthesized from constituent amino acids by the
sequential action of .gamma.-glutamylcysteine synthetase
(.gamma.-GCS) and GSH synthetase (GS) where .gamma.-GCS is rate
limiting. GSH plays a major role in cellular defenses against
oxidative stress and reactive electrophiles. GSH also participates
in the reductive detoxification of hydrogen peroxide and lipid
peroxides. Each of these reactions leads directly or indirectly to
the formation of glutathione disulfide (GSSG), a species that is
reduced intracellularly to GSH by glutathione reductase (GR) in a
NADPH-dependent reaction. GR normally maintains the total
glutathione pool in a predominately-reduced state; thus, redox
cycling between GSSG and GSH does not usually have a major
influence on cellular GSH levels. Extracellular degradation of GSH
and GSSG is carried out rapidly by membrane bound
.gamma.-glutamyl-transpeptidase (GGT) and cysteinyl-glycine
dipeptidase (DP). In the lung, GGT is primarily located on the
airspace epithelial surface and facilitates .gamma.-glutamyl
absorption from ELF GSH. Although GSH reacts spontaneously with
some electrophiles, most of these reactions require catalysis by a
family of enzymes known as GSH S-transferases (GST). The initial
products are chemically stable sulfides of GSH, but upon further
metabolism form S-substituted L-cysteines that are acetylated to
form mercapturic acids and readily excreted in the urine. Cellular
synthesis, metabolism and transport of GSH are summarized in FIG.
14.
Important Roles of GSH
[0131] Bronchoalveolar lavage (BAL) leukocytes from CF patients
have exaggerated cytokine release and oxidant generation responses
to stimuli and CF patients aerosolized with GSH had suppressed
oxidant generation from stimulated BAL leukocytes.
[0132] Many CF patients are chronically infected with Pseudomonas
aeruginosa, which releases redox active pigments that generate
oxidants, inhibit anti-proteases and induce neutrophil apoptosis.
GSH is a major water-soluble anti-oxidant in the ELF that protects
anti-proteases from inactivation by oxidants and prevents excessive
tissue destruction from neutrophil derived proteases like
neutrophil elastase. This scenario creates an imbalance between
antiproteases and proteases in the lung leading to increased tissue
destruction.
Prevention of Oxidative Stress in the Mitochondria by Superoxide
Dismutase and Glutathione
[0133] In FIG. 1 during oxidative phosphorylation, the mitochondria
generates superoxide anion (O.sub.2..sup.-. and hydrogen peroxide
(H.sub.2O.sub.2) through the respiratory chain. Superoxide
formation arises from two sites along the respiratory chain: the
NADH dchyrgrogenase (I) and ubiquinone Q-cytochrome b complex
(III). Normally, 2-4% of the electron flux through the respiratory
chain reduces oxygen to O.sub.2..sup.- instead of water. Once
formed, O.sub.2..sup.- is rapidly reduced to H.sub.2O.sub.2 by
manganese superoxide dismutase (MnSOD). H.sub.2O.sub.2 may react
with reduced iron (Fe.sup.+2) to form the highly toxic hydroxyl
radical (OH.), or detoxified to H.sub.2O by the action of
glutathione peroxidase (GPx). GPx consumes reduced glutathione
(GSH) in this catalysis to form oxidized glutathione (GSSG). GSH is
regenerated from the GSSG by glutathione reductase (GRx). GSH
synthesis, however, occurs in the cytoplasm and requires transport
into the mitochondria. Little is currently known about the
transporter(s) responsible for regulating mitochondrial GSH.
[0134] The extracellular reducing environment is critical for
proper immune function such as antigen presentation and subsequent
T cell proliferation. GSH can affect the nature and level of
antigen presentation in APCs by altering the protein disulfide
bonds required for proteolytic digestion of the antigen. GSH can
also regulate cytokine release, such as IL-4, from lymphocytes and
directly suppress inflammatory responses. GSH is a potent mucolytic
agent due to its ability to cleave disulfide bonds. The thickened
mucus can lead to airway obstruction and decreased bacterial
clearance.
[0135] GSH may be an important reactant with nitric oxide and may
regulate nitric oxide's bioavailability and influence whether
nitric oxide acts as an antioxidant or prooxidant. GSH is also a
precursor of S-nitrosoglutathione (another important monothiol)
that is an endogenous bronchodilator and found to be deficient in
lung of CF patients.
[0136] The normal healthy adult human liver synthesizes 8-10 grams
of GSH daily. Normally, there is an appreciable flow of GSH from
liver into plasma. The intracellular level of GSH in mammalian
cells is in the range of 0.5-10 millimolar, while micromolar
concentrations are typically found in blood plasma. Intracellular
glutathione is normally over 90% found in a reduced form (GSH). The
major organs involved in the inter-organ transport of GSH are the
liver and the kidney, which is the primary organ for clearance of
circulating GSH. It has been estimated to account for 50-67% of net
plasma GSH turnover. Several investigators have found that during a
single pass through the kidney, 80% or more of the plasma GSH is
extracted, greatly exceeding the amount that could be accounted for
by glomerular filtration. While the filtered GSH is degraded
stepwise by the action of the brush-border enzymes
y-glutamyltransferase and cysteinylglycine dipeptidase, the
remainder of the GSH appears to be transported via an unrelated,
Na+-dependent system present in basal-lateral membranes.
[0137] Glutathione exists in plasma in four forms: reduced
glutathione (GSH), oxidized glutathione (GSSG), mixed disulfide
with cysteine (CySSG) and protein bound through a sulfhydryl
linkage (GSSPr). The distribution of glutathione equivalents is
significantly different than that of cyst(e)ine, and when either
GSH or cysteine is added at physiological concentration, a rapid
redistribution occurs. In erythrocytes, GSH has been implicated in
reactions that maintain the native structure of hemoglobin and of
enzymes and membrane proteins. GSH is present in erythrocytes at
levels 1000 times greater than in plasma. It functions as the major
small molecule antioxidant defense against reactive oxygen
species.
[0138] The importance of thiols and especially of GSH to lymphocyte
function is known. Adequate concentrations of GSH are required for
mixed lymphocyte reactions, T-cell proliferation, T- and B-cell
differentiation, cytotoxic T-cell activity, and natural killer cell
activity. Adequate GSH levels have been shown to be necessary for
microtubule polymerization in neutrophils. Intraperitoneally
administered GSH augments the activation of cytotoxic T-lymphocytes
in mice, and dietary GSH was found to improve the splenic status of
GSH in aging mice, and to enhance T-cell-mediated immune
responses.
[0139] Decreasing GSH by 10-40% can completely inhibit T-cell
activation in vitro. Depletion of intracellular GSH has been shown
to inhibit the mitogenically-induced nuclear size transformation in
the early phase of the response. Cysteine and GSH depletion also
affects the function of activated T-cells, such as cycling T-cell
clones and activated cytotoxic T-lymphocyte precursor cells in the
late phase of the allogenic mixed lymphocyte culture. DNA synthesis
and protein synthesis in IL-2 dependent T-cell clones, as well as
the continued growth of preactivated CTL precursor cells and/or
their functional differentiation into cytotoxic effector cells are
strongly sensitive to GSH depletion.
[0140] The nucleoplilic sulfur atom of the cysteine moiety of GSH
serves as a mechanism to protect cells from harmful effects induced
by toxic electrophiles. The concept that glutathione S-conjugate
biosynthesis is an important mechanism of drug and chemical
detoxification is well established. GSH conjugation of a substrate
generally requires both GSH and glutathione-S-transferase activity.
The existence of multiple glutathione-S-transferases with specific,
but also overlapping, substrate specificities enables the enzyme
system to handle a wide range of compounds.
[0141] Because of its known role in renal detoxification and its
low toxicity, GSH has been explored as an adjunct therapy for
patients undergoing cancer chemotherapy with nephrotoxic agents
such as cisplatin, in order to reduce systemic toxicity. Other
studies have shown that i.v. GSH coadministration with cisplatin
and/or cyclophosphamide combination therapy, reduces associated
nephrotoxicity, while not unduly interfering with the desired
cytotoxic effect of these drugs.
[0142] GSH functions in many important biological phenomena,
including the synthesis of proteins and DNA, transport, enzyme
activity, metabolism, and protection of cells from free-radical
mediated damage. GSH is one of the primary cellular antioxidants
responsible for maintaining the proper oxidation state within the
body. GSH is synthesized by most cells, and is also supplied in the
diet.
Biochemical Consequences of Oxidative Stress
[0143] FIG. 2 illustrates a schematic flow of reactive oxygen
species (ROS; e.g., superoxide, hydrogen peroxide, hydroxyl
radical) and reactive nitrogen species (RNS; e.g., nitric oxide,
nitrogen dioxide, peroxynitrite) on macromolecules. These species
may oxidize or nitrate proteins, lipids and DNA. Protein oxidations
may result in the loss of function (e.g., sulfhydryl and tyrosine
oxidation) and protein cross-linking. Lipid oxidation generates
hydroperoxides and other oxidants (e.g., alkoxyl and alkylperoxyl
radicals) that can propagate the oxidative stress. DNA oxidations
cause strand breaks, base modifications and strand cross-linking.
Ultimately, these biochemical alterations may lead to tissue
injury.
[0144] In certain embodiments, the transport of thiol-containing
compounds for example GSH may be increased in order to replenish
inadequate supplies. In other embodiments, the intracellular
distribution of thiol-containing compounds may be targeted in order
to redistribute GSH to such cell compartments for example the
mitochondria. Because of the importance of glutathione in
preventing this cellular oxidation, glutathione is continuously
supplied to the tissues. However, under certain conditions, the
normal, physiologic supplies of glutathione are insufficient,
distribution inadequate or local oxidative demands too high to
prevent cellular oxidation. Under certain conditions, the
production of and demand for glutathione are mismatched, leading to
insufficient levels. In other situations, certain tissues or
biological processes consume glutathione so that the intracellular
levels are inadequate. In either case, by increasing the levels of
glutathione, increased amounts may be directed into tissues.
Direct GSH Applications
[0145] It is believed that beneficial physiological effects of
orally administered glutathione are difficult or impossible to
achieve, or the efficiency is so low as to make supplementation by
this route unproductive. The protocols for oral administration of
glutathione were not optimized and therefore the bioavailability of
the glutathione was unassured and variable. All prior
pharmaceutical attempts by others to safely, effectively and
predictably raise intracellular GSH through oral therapy with GSH
have met with very little success. It was also believed that orally
administered glutathione would tend to be degraded in the stomach,
and that it is particularly degraded under alkaline conditions by
desulfurases and peptidases present in the duodenum.
[0146] Because of the poor or variable results obtained with GSH,
orally absorbed pro-drugs and precursors have also been used. A
known pharmacological regimen provides intravenous glutathione in
combination with another agent, such as cis-platinum (a free
radical associated metal drug), doxorubicin, or daunorubicin (free
radical associated drugs which interact with nucleic acid
metabolism), which produced toxic side effects related to free
radical reactions. The combination of the components has revealed
limited success.
[0147] Although the parenteral infusion of cysteine precursors as
well as glutathione esters is believed to be an effective way to
increase or maintain a sufficient level of intracellular
glutathione, it would of course be desirable if the intracellular
glutathione level could be maintained or increased through an
enteral diet. One of the difficulties in increasing through an
enternal regimen intracellular glutathione levels is that it is not
typically possible merely to provide an enteral amino acid solution
rich in cysteine. Cysteine typically will crystallize out as
cystine in solution, e.g., an amino acid solution. Cystine is not
readily biologically available to cells. Therefore, cysteine is not
biologically available as a pharmaceutical.
[0148] Other proposed administration of glutathione is using
aerosol administration through the nasal passageway. This route
also proved fruitless since the glutathione cannot penetrate the
mucosal layer very efficiently and it is often oxidized prior to
reaching the intended area (i.e. namely the ELF of cystic fibrosis
patients)(Buhl, R. PNAS 87:4063, 1990 and Roum J. of Physiol.
87:438 1999).
[0149] In one embodiment, the transport of existing GSH out of
cells or tissue will be increased via transporters. In other
embodiments, "GSH-like" mono-thiol compound secretion out of cells
or tissue will be increased. In other embodiments, the
intracellular distribution of thiol-containing compounds may be
altered in order to prevent or treat a condition. In one
embodiment, one or more compounds may be used to restore
thiol-containing compounds extracellularly. In other embodiments,
one or more compounds may be used to restore GSH levels
extracellularly. In one embodiment, one or more compounds may be
used to restore thiol-containing compounds intracellularly. In
other embodiments, one or more compounds may be used to restore GSH
levels intracellularly.
[0150] In other embodiments, other mono-thiol containing compounds
that can be exported are cysteinyl leukotriene, LTC4 and any other
mono-thiol containing compounds capable of excretion by one or more
ABC transporters described below.
Thioredoxin
[0151] Thioredoxin (TXR) is a potent protein disulfide reductase
found in most organisms that participates in many thiol-dependent
cellular reductive processes. Along with glutathione, thioredoxin
is also a major small molecular weight thiol-containing compound
synthesized de novo in mammalian cells. In addition to its ability
to effect the reduction of cellular proteins, thioredoxin can act
directly as an antioxidant (e.g it scavenges free radicals) or can
increase the oxidative stress in a cell by autooxidizing (e.g.
generating superoxide radicals through autoxidation). Thioredoxin
can also directly induce the production of MnSOD (manganese
superoxide dismutase, sod2).
[0152] Investigators have reported the use of thioredoxin to treat
several conditions. One invention taught that "thioredoxin
compounds" can be topically applied to the eye to reduce disulfide
bonds of oxidized lens proteins involved in cataract formation,
thus preventing or reducing a cataractous condition (U.S. Pat. No.
4,771,036). Other investigators have reported the intravenous
injection of thioredoxin to treat post-ischemia tissue injury in
rats or dogs (Fukuse, et al., pp. 387-391, 1995, Thorax, Vol. 50;
Yagi et al., pp. 913-921, 1994, J. Thorac. Cardiovasc. Surg., Vol.
108). These studies measured only the physiological effects of
thioredoxin on ischemia and suggested that thioredoxin had limited
success and was acting as an antioxidant (scavenger of free
radicals). In these examples, thioredoxin was intravenously
administered in these studies, and was only present for a very
short time at the site of damage, if at all. It is further unknown
whether thioredoxin even went to the specific site of damage, the
lung. Such reports do not disclose or suggest a method or
composition to increase a thiol-containing compound transporter(s)
having a distinct ability to actively pump the existing
thiol-containing compounds to the site of interest (i.e. the ELF of
the lung).
[0153] Investigators have found thioredoxin in most organisms and
it participates in many thiol-dependent cellular reductive
processes. In humans, thioredoxin is also referred to as adult T
cell leukemia-derived factor (ADF). Intracellularly, most of this
ubiquitous low molecular weight (11,700) protein remains reduced.
Reduced or oxidized thioredoxin can enter intact cells. It has two
vicinal cysteine residues at the active site that in the oxidized
protein forms a disulfide-bridge located in a protrusion from the
protein's three-dimensional structure. The flavoprotein thioredoxin
reductase catalyzes the NADPH-dependent reduction of this
disulfide. Small increases in the presence of thioredoxin can cause
profound changes in sulfhydryl-disulfide redox status in proteins.
Thus, thioredoxin is extremely potent as a reducing agent.
Extremely low concentrations of thioredoxin are effective in
reducing disulfides in insulin, fibrinogen, human chorionic
gonadotropin, blood coagulation factors, nitric oxide synthase,
ribonucleotide reductase, glucocorticoid receptors and other
proteins. The rate of reduction of insulin disulfide by thioredoxin
has been found to be 10,000 times higher than that by DTT
(dithiothreitol). Thioredoxin has also been found to be a greater
reducer than GSH as well. Thus, reduced thioredoxin is an extremely
potent protein disulfide reductase. A preferred embodiment of this
invention comprises the increase of thiol-containing compound
transporters to increase the export of thioredoxin. In other
embodiments, these transporters are used to increase the export of
thioredoxin into the ELF of the lung epithelium by apical
exportation.
[0154] Trx together with thiorcdoxin reductase (TrxR) and NADPH
comprise the thioredoxin system. Thioredoxins have a redox-active
disulfide/dithiol and are reduced by selenium-dependent thioredoxin
reductases with a Gly-Cys-Sec-Gly active site. Thioredoxins in the
cytosol, mitochondria or extracellularly are the cells major
disulfide reductases required for the control of redox potential
and signalling by thiol redox control.
Glutaredoxin
[0155] Glutaredoxin (Grx) catalyzes disulfide oxidoreductions
involving glutathione (GSH) and the glutaredoxin system comprises
Grx, GSH, glutathione reductase and NADPH. Glutaredoxins which have
a classical Cys-Pro-Tyr-Cys active site and a. binding site for GSH
are required for GSH to operate in thiol-dependent reductions like
the synthesis of deoxyribonucleotides by ribonucleotide reductase.
Glutaredoxins play a specific role in redox regulation via
reverible glutathionylation of proteins where both the synthesis
and degradation of the mixed disulfide with glutathione is
catalysed by multiple dithiol or monothiol glutaredoxins (Amer, E S
J and Holmgren, A. (2000), Eur. J. Biochem., 267, 6102-6109; Zhao,
R., Masayasu, H. and Holmgren, A. (2002) Proc. Natl. Acad. Sci.
USA, 99, 8579-8584; Lundberg, M. et al. (2002) J. Biol. Chem. 276,
26269-26275.). Glutaredoxin is characterized by a dithol/disulphide
redox-active site. Human glutaredoxin, unlike E. coli glutaredoxin,
has an additional pair of cysteine residues that may play a
regulatory role in its activity. One embodiment includes using one
or more compounds to increase the extracellular transport of
glutaredoxin.
ABC Superfamily
[0156] The ABC superfamily transport proteins to a variety of
molecules, ranging from ions to proteins, across cell membranes.
(For a review see C. F. Higgins, Ann. Rev. Cell Biol. 8, 67 (1992)
and Klein, I. Biophys.Biochem. Abstracts 1461: 237 (1999)). There
are approximately 50 known ABC transporters in the human. As
mentioned earlier, 13 genetic diseases associated with defects in
14 of these transporters exist.
Mechanisms in Multi-Drug Resistance: ABC-Transporters and
Detoxification Enzymes
[0157] The cellular components responsible for the phenomenon of
multi-drug resistance (MDR) in the structural context of for
example, hepatocytes include intrinsic transmembrane proteins,
generically called drug efflux pumps, and the enzymes responsible
for detoxification and biotransformation of xenobiotics (i.e.
pesticides). The transporters, which have been called
P-glycoprotein (MDR), multidrug resistance related protein (MRP)
and GS-X pump and which are believed to be involved in the primary
active pumping of xenobiotics from the cells, are now known as the
ATP-binding cassette (ABC) transporters. The major drug efflux
pumps are in the superfamily of ABC transporters. Of these
ABC-ATPases, the major subfamilies are the MDR/TAPs and MRPs
(multi-drug resistance related proteins). The natural substrates
are indicated for the drug efflux pumps in the references indicated
(Shabbits, J. A., et al., Molecular and pharmacological strategies
to overcome Multidrug resistance. Expert Rev. Anticancer Ther., 1,
585-594 (2001); Di, P. A., et al., Modulation by flavonoids of cell
multidrug resistance mediated by P-glycoprotein and related ABC
transporters. Cell. Mol. Life. Sci., 59, 307-322 (2002); Meier, P.
J., and Stiger, B., Bile salt transporters. Annu. Rev. Physiol.,
64, 635-661 (2002)). One ABC transporter family example, the
P-glycoproteins (P-gp), transport chemotherapeutic drugs. This
family includes the CFTR, which controls chloride ion fluxes, as
well as insect proteins that mediate resistance to anti-malarial
drugs. P-glycoprotein is believed to confer resistance to multiple
anticancer drugs by acting as an energy dependent efflux pump that
limits the intracellular accumulation of a wide range of cytotoxic
agents and other xenobiotics. In addition, other compounds that are
excluded from mammalian cells by P-glycoprotein are frequently
natural product-type drugs such as thiol-containing compounds but
also other large heterocyclic molecules are also "substrates" for
this efflux pump. In one embodiment, a compound may be used to
modulate the extracellular transport of thiol-containing compounds
by increasing the activity of CFTR.
Domain Organization of Multi-Drug Related Protein (MRP1)
[0158] MRP1 is a representative of the second major subfamily, MRP,
of multidrug ABC transporters. MRPs have an extra TMD
(transmembrane domain) and have the general form of
TMD.sub.0(ABC-TMD).sub.2. FIG. 13 shows the transporter as an
intrinsic membrane protein (Kruh, G. D., et al. MRP subfamily
transporters and resistance to anticancer agents. J. Bioenerg.
Biomembr.33, 493-501 (2001); Borst, P., et al. A family of drug
transporters: the multidrug resistance-associated proteins. J.
Natl. Cancer Inst., 92, 1295-1302 (2002); Rosenberg, M. F., et al.
The structure of the multidrug resistance protein 1 (MRP1/ABCC1),
crystallization and single-particle analysis. J. Biol. Chem., 276,
16076-16082 (2001)).
[0159] Also, MRP2 (multi-drug resistance protein) is known to
transport GSSG and glutathione conjugates. MRP1 is ubiqutiously
expressed in normal tissues and is a primary active transporter of
GSH, glucuronate and sulfate conjugated and unconjugated organic
anions of toxicological relevance (i.e. herbicides, mycotoxins,
heavy metals, natural products). The most studied of these proteins
is the basolaterally expressed MRP-1 that co-transports GSH with
natural product toxins such as aflatoxin and vincristine. In the
liver, MRP-2 functions as a low-affinity export pump for release of
GSH across apical domains
[0160] Direct support indicates that the CFTR protein is involved
in only half of the GSH transport into the pulmonary ELF. However,
the transporter(s) for the other half of the glutathione are
currently not known. The embodiments target other ABC cassette
protein super family members that contribute to the apical
transport of glutathione in tissues (i.e. the lung) and these
transporters could be manipulated to endogenously restore
glutathionc to the lung ELF (Table 2).
[0161] Many of these ABC transporters function on the apical
membrane of epithelial cells (i.e. P-gp and MRP2 etc.) thus
enabling the export of several components. The expression of these
transporters appears to be tissue specific. For example, MRP2 is
found almost exclusively in apical membranes of polarized cells
(i.e. kidney, liver, lungs and the intestine). MRP1 is located in
the basolateral side of epithelial cells (Laouari, D. et. al. "Two
Apical drug transporters, P-gp and MRP2, are differently altered in
chronic renal failure" AJP--Renal Physiology, 280 (4):F636-F645,
April 2001). MRP1 and related transporters MRP2 and MRP3 have
overlapping substrate specificities but there tissue distribution
varies. Thus, several embodiments are directed at the increase in
thiol-containing compounds excretion via transporters localized to
a specific tissue. In other embodiments, these thiol-containing
compound transporters include increasing thiol-containing compound
excretion via transporters localized in the lung. Additional
embodiments include thiol-containing compound transporters to
increase thiol-containing compound excretion via transporters
localized in the lung epithelia. In still other embodiments, these
transporters are transporters found in the pancreas,
gastointestinal tract, sweat glands, the vas deferens, and
kidney.
[0162] P-glycoprotein has been identified in a variety of tumor
types. This information spurred on the search for compounds that
are capable of blocking its function and consequently, reversing
resistance to the anti-cancer agents. A large number of agents
called chemosensitizers or reversing agents have been identified.
Chemosensitizers that can reverse P-glycoprotein-mediated multidrug
resistance include verapamil and cyclosporin A. These agents
interfere with the ability of the transport system to excrete the
chemical agent.
[0163] One embodiment includes a method to identify compounds that
increase the excretion of thiol-containing compounds that normally
"ride along" with the chemotherapy agent as the drug is excreted
from the cell. The mode of excretion is often associated with the
same ABC transporters that excrete chemotherapy drugs in resistant
tumor cells. Other embodiments detail the use of some of these
chemotherapy drugs to increase the excretion of the
thiol-containing compounds by affecting the transporters. Some of
these embodiments detail the excretion of mono-thiol compounds
(i.e. glutathione, cysteine etc.). Other embodiments detail the
secretion of di-, tri- and multi-thiol-containing compounds (i.e.
thioredoxin, gluteradoxin) using some of the same chemotherapy
drugs.
[0164] In other embodiments of this invention, inhibitors of
compounds that negatively affect ABC transporter activity,
expression and/or synthesis will be used. In other embodiments,
inhibitors of various forms of p53 that are known to suppress
SP1-DNA binding activity will be used to increase the activity of
the transporters of the thiol-containing compounds. SP1-DNA is
thought to stimulate the expression of the ABC transporters. (Iida,
T. et. al. Cancer Gene Therapy 2001 October; 8(10):803-814.)
[0165] Several agents are currently known to modulate the activity
and or expression of ABC transporters and the ability of these
transporters to excrete chemotherapy drugs. Some embodiments of
this invention include the use of agents to increase the presence
or the activity of the transporters to deliver thiol-containing
compounds "along with" chemotherapy drugs. One embodiment relates
to the use of dexamethasone, shown to increase the level of
glutathione in the lung ELF .see FIG. 17. The increase in the P-gp
by dexamethasone is rapid, peaking at Dayl-3. (DeMeule, M. et. al.
FEBS Letters 442 (1999) 208-214). Using a single dose, cisplatin
(cis-dichlorodiammne platinum (II) induces P-gp 200-300.times. in
the renal basement membrane, liver and intestine (DeMeule, M. Am.
J. Physiol. 227 (Renal Physiol.46): F832-F840, 1999). Other
embodiments include the use of cis-platin to induce the presence of
ABC-transporters for increasing the excretion of thiol-containing
compounds. Additional embodiments include the use of cis-platin to
increase the presence of specific transporter such as P-gp
transporters for increasing the excretion of thiol-containing
compounds. Other embodiments include the use of vinblastine to
induce the expression of MRP2; daunorubicin to induce MRP1
expression and sulfinpyrazone at low doses to induce the
co-transport of GSH and sulfinpyrazone in a "positive
cooperativity" mode.
[0166] Sulfasalazine is a well-known drug that is used to treat
rheumatoid arthritis and inflammatory bowel disease. However, the
mechanism of action of sulfasalazine in these diseases is poorly
understood. It has been proposed that sulfasalazine possesses
anti-inflammatory properties including the inhibition of NFkB
(Pittet I F, Lu L N, Morris D G, Modelska K, Welch W I, Carey H V,
Roux J, and Matthay M A. Reactive nitrogen species inhibit alveolar
epithelial fluid transport after hemorrhagic shock in rats. J.
Immunol. 166:6301-6310, 2001), lymphocyte x(c)-cystine transporter
(Gout P W, Buckley A R, Simms C R, and Bruchovsky N. Sulfasalazine,
a potent suppressor of lymphoma growth by inhibition of the
x(c)-cystine transporter: a new action for an old drug. Leukemia
15:1633-1640, 2001), and selective modulation of B cell function
(Hirohata S, Ohshima N, Yanagida T, and Aramaki K. Regulation of
human B cell function by sulfasalazine and its metabolites. Int
Immunopharmacol 2:631-640, 2002). Another possible mechanism for
the anti-inflammatory actions of sulfasalazine is it and its
metabolite (p-amino salicylic acid) ability to increase glutathione
efflux in epithelial cells (see table 3). In one embodiment,
sulfasalazine may be used alone or in combination with one or more
additional agents to increase the transport of thiol-containing
compounds from one or more cells. In another embodiment,
sulfasalazine may be used alone or in combination with one or more
additional agents to increase the transport of thiol-containing
compounds from one or more lung cells. In still another embodiment,
sulfasalazine may be used alone or in combination with one or more
additional agents to increase the transport of thiol-containing
compounds (for example, glutathione) to the lung ELF.
[0167] Other agents known to increase the expression of multi-drug
resistance transporters are naturally occurring substances such as
berberine, an alkaloid of the Chinese herb referred to as
Goldenseal. Berberine has been shown to increase the expression of
P-gp (pgp-170) in hepatoma cells of humans and mice (Lin, H L
"Up-regulation of multidrug resistance transporter expression by
berberine in human and murine hepatoma cells," Cancer 1999 May 1;
85(9):1937-42). Therefore, other embodiments of this invention
include the use of naturally occurring substances extracted from
herbs to increase the expression and/or activity of transporters of
thiol-containing compounds. Further, other embodiments include the
use of Goldenseal extracts to increase the expression and/or
activity of transporters of thiol-containing compounds. In
addition, embodiments include the use of berberine to increase the
expression and/or activity of transporters of thiol-containing
compounds. Exposure to microorganisms can also increase the lung
expression of both CFTR and MRP-2 and this correlates with a 6-fold
increase in ELF glutathione levels (see FIG. 18).
[0168] Still other agents increase the expression of MRPs and other
ABC transporters namely, xenobiotics. The very chemicals that are
excreted from cells to protect the cell from their exposure also
induce the transporters. This has been documented in mammals as
well as aquatic life (Bard et al. "Expression of P-glycoprotein and
cytochrome p450 1A in intertidal fish (Anoplarchus) exposed to
environmental contaminants." Aquat Toxicol. 2002) October 2; 60
(1-2): 17-32). b other embodiments, xenobiotics or xenobiotic-like
compounds such as a sufficient amount of pesticides or other
xenobiotics may be used to increase the expression and/or activity
of transporters of thiol-containing compounds.
[0169] Other compounds that may affect the level of ABC
transporters are MMPs (matrix metalloproteinases). The MMPs are
members of a family of at least 20 proteolytic enzymes that contain
a zinc ion at their active sites and can degrade collagen,
elastins, and other components of the extracellular matrix (ECM).
Cytokine activation of cells can lead to increased processing of
MMPs from inactive zymogens to the active enzymes. Cytokines and
their receptors can also be substrates for MMP action. Many of the
membrane-bound cytokines, receptors, and adhesion molecules can be
released from the cell surface by the action of a subset of
metalloproteinases called convertases or adamalysins. This may be
one mechanism for the down-regulation of cell surface receptors and
transporters such as ABC transporters. (Nagase, H., and Woessner,
J. F., Jr., Matrix metalloproteinases. J. Biol. Chem., 274,
21491-21494 (1999). Rooprai, H. K., et al., The effects of
exogenous growth factors on matrix metalloproteinase secretion by
human brain tumour cells. Br. J. Cancer, 82, 52-55 (2000); Stone,
A. L., et al., Structure-function analysis of the ADAM family of
disintegrin-like and metalloproteinase-containing proteins
(review). J Protein Chem., 18, 447-465 (1999); Killar, L., et al.,
Adamalysins. A family of metzincins including TNF-a converting
enzyme (TACE). Ann. NY Acad. Sci., 878, 442-452 (1999)).
[0170] Collagen is an intrinsic component of the extracellular
matrix in the lung and is continuously being synthesized and
degraded. The majority of collagen is synthesized and secreted by
fibroblasts and lung alveolar macrophages secrete matrix
metalloproteinases (MMPs) that degrade it. The activity of MMPs is
kept in check by the release of tissue inhibitors of
metalloproteinases (TIMPs). During inflammation the balance between
MMPs and TIMPs is disrupted and is thought to lead to enhanced
matrix destruction, cytokine inactivation, and shedding of cell
surface molecules, which can lead to amplification of the
inflammatory response. The redox state of the extracellular spaces
in the lung is set by glutathione and regulates the balance of MMP
and TIMP activities.
[0171] One embodiment includes the use of MMP inhibitors for
exampleTIMP1, TIMP2, or TIMP3 to inhibit MMPs that may result in
the increase in availability of ABC transporters.
Knockout Mice
[0172] Several mouse strains have been used to characterize the
function of ABC transporters in certain systems such as Congenic
C57BL/6J-CFTR.sup.TM1UNC
[0173] The CFTR KO has an increased inflammatory response and
mortality towards Pseudomonas aeruginosa (strain M57-15) infection.
(Van Heeckeren et al. J. Clin. Invest. 100:2810, 1997). FABP-hCFTR
gut corrected C57BL/61Cftr.sup.tm1Unc KO mice. These CFTR KO mice
can survive on normal diet without intestinal obstruction. These
strains allow one to directly compare effects of CFTR on epithelial
function in the same animal (i.e. lung vs intestine)(Zhou et al.
Science 266:1705, 1994, Steagall et al. Am. J. Respir. Cell Mol.
Biol. 22:45, 2000).
[0174] The CFTR KO (knock-out) mouse does not totally recapitulate
CF lung disease but its lungs are not normal and it provides a
valuable animal model to study the mechanisms by which the CFTR
gene defect directly contributes to the GSH imbalance. Previous
data shows there is a 50% decrease in the lung ELF glutathione
concentration. The CFTR KO mouse is useful for determining whether
this GSH imbalance plays a role in the exaggerated inflammatory
responses to oxidative stress and altered host defense. Another
secondary observed condition of the CFTR KO mice is increased
oxidation of lung DNA and lipids likely due to low GSH levels.
[0175] CFTR KO mice may be used to separate out CFTR's contribution
from the other transporters. Many of the ABC transporter genes have
been cloned and sequenced and commercial antibodies are readily
available for most members of this family due to their interest by
cancer researchers as markers for tumor resistance to
chemotherapeutics. The use of this extensive database localization,
gene expression and function of these proposed transporters in the
lung. Inducers of these apical transporters will be assessed by
changes in GSH levels (namely bronchoalveolar lavage fluid (BALF)
GSH of the lungs). The correlation of this data will determine
which of the apical ABC transporters that are critical in
regulating ELF GSH levels and may be targets for restoring ELF GSH
in the CF lung or other tissues.
[0176] Since CFTR modulates only 50% of glutathione transport,
other transporters were implicated in the excretion of the
remaining glutathione. Thus, other embodiments comprise targeting
"non-CFTR" transporters for modulating the excretion of
thiol-containing compounds. Still other embodiments specifically
aim to increase the thiol-containing compound activity of these
"non-CFTR" transporter systems. More specifically, other
embodiments aim to increase the thiol-containing compound excretion
activity of "non-CFTR" ABC-transporters. In addition, other
embodiments aim to increase the thiol-containing compound excretion
activity of MRP-1, MRP-2 and/or MDR-1.
[0177] FIG. 3 illustrates defects in the Cystic Fibrosis
Transmembrane Regulator Protein (CFTR) may lead to obstructive lung
disease and fibrosis. A multitude of mutations in the cystic
fibrosis (CF) gene can result in a CFTR deficiency or a defective
CFTR. Whether because of a deficiency or a defect in CFTR,
alterations in the secretion of Cl.sup.- and/or glutathione lead to
a perpetuation of infection and inflammation in the lung.
Ultimately, this ongoing cycle of infection and inflammation leads
to obstructive lung disease, fibrosis and death. BALF and lung
tissue may be analyzed for evaluation of the presence or absence of
GSH and evaluation of host defense in an anti-Pseudomonas
assay.
Cell Lines
[0178] Several cell lines may be used in the embodiments for
example; CRL-1687, HTB-79, and the A549 to test for potential
agents that stimulate thiol-containing compound transport. Both the
CRL-1687 and HTB-79 cell lines are derived from human pancreatic
adenomas. The primary difference between these two cell lines is
their expression of the cystic fibrosis transmembrane regulator
protein (CFTR). HTB-79 cells express CFTR while the CRL-1687 cells
do not. This difference in CFTR expression provides a method for
identifying potential CFTR-dependent mechanisms in experiments
where the two lines are exposed to identical conditions but yield
differing results. In these investigations, the A549 cells
represent secretory cells of the lung epithelium. A549 cells can be
grown in a two-compartment culture system that produces separate
apical and basolateral compartments that facilitate the
identification of apical transport stimulators.
HTB-79 Cells.
[0179] Purchased from American Type Culture Collection (ATCC) at
passage eighteen. This cell line is derived from a human
adenocarcinoma of the pancreas. Similar to normal pancreatic cells,
the HTB-79 cells constitutively express CFTR. These cells may be
grown in Iscove's modified Dulbecco's medium supplemented with 20%
fetal bovine serum. Penicillin (100 U/mL) and streptomycin (100
U/mL) were added to prevent bacterial contamination
CRL-1687 Cells
[0180] CRL-1687 cell are derived from a human adenocarcinoma of the
pancreas that does not express the CFTR protein. The cells were
grown in complete growth medium RPMI 1640 supplemented with 10%
fetal bovine serum. Penicillin (100 U/mL) and streptomycin (100
U/mL) were added to prevent bacterial contamination.
A-549 Cells
[0181] A-549 cell may be purchased from ATCC at an unknown passage.
This cell line is derived from a lung carcinoma. The cells are
maintained in Ham's F12K medium supplemented with 10% fetal bovine
serum. Penicillin (100 U/mL) and streptomycin (100 U/mL) were added
to prevent bacterial contamination.
[0182] A widely used cell line in experimental studies of certain
types of cancer is NCI-H69 (H69) (Gazdar et al., Cancer Res. 40,
3502-3507 (1980)) (ATCC HTB 119). This cell line was repeatedly
exposed to an anthracycline, such as daunorubicin or epirubicin and
preferably doxinibicin (DOX), and selected to produce a "multidrug
resistant cell line", designated as H69AR. A description of the
procedures that can be used to produce a multidrug resistant cell
line such as H69AR is found in Cole, Cancer Chemother Pharmacol.
17, 259-263 (1986) and in Mirski et al., Cancer Research 47,
2594-2598 (1987).
[0183] The H69AR cell line (ATCC CRL 11351) is about 50-fold
resistant to DOX as compared to the parental H69 cell line. H69AR
is also cross-resistant to a wide variety of natural product-type
drugs. On the other hand, drugs such as carboplatin, 5-fluorouracil
and bleomycin are equally toxic to both sensitive H69 and resistant
H69AR cells. Although the cross-resistance pattern of H69AR cells
is typical of resistance associated with increased levels of P-gp,
these cells are different in that they display little or no
collateral sensitivity to hydrophobic drugs such as steroids or
local anaesthetics. Another distinguishing feature of H69AR of
potential clinical relevance that distinguishes it from P-gp
overexpressing cell lines is the limited ability of verapamil,
cyclosporin A and other chemosensitizing agents that interact with
P-gp, to reverse DOX resistance in these cells. The absence of P-gp
overexpression supports the suggestion that H69AR provides a
clinically relevant model of drug resistance in lung cancer as well
as a model for the overexpression of a thiol-containing compound
transporter that is not P-gp.
[0184] In one embodiment, a cell line may be used to assay for a
substance that increases the thiol-containing compound excretion
and/or affects the thiol-containing compound transporter itself.
Cells from a cell line may be incubated with a test agent (i.e. a
flavone, isoflavone, flavanone etc.) suspected of affecting the
thiol-containing compound excretion. Analyzing the amount of
thiol-containing compound excretion into an extracellular medium
and comparing these results to a control (parental cell line) can
determine the effect of an agent on the transporter.
[0185] The doses of agent (stimulatory) are estimated from the
literature, but will be titrated to determine doses necessary to
affect the thiol-containing compound transporter (i.e. lung MRP-2
and MDR-1). In one embodiment, fluorescent dyes may also be
transported by the thiol-containing compound transporters (i.e.
MDR-1 and MRP-2), such as rhodamine 123 and calcein AM and may be
measured in the area under examination (i.e. lung ELF). Thus, the
activity of the transporter can be measured by measuring the amount
of dye transported.
[0186] In one embodiment, a substance that is suspected of
increasing the excretion of thiol-containing compounds can be
identified. Therefore, it is possible to use this method to
identify substances that may be useful in the treatment of
thiol-containing compound excretion deficient conditions. At least
one of the following compounds for example a flavone, an
isoflavones, a flavanones, a flavanols, a benzoic acid derivative,
an indole derivative, a 1,4-naphthoquinone, a 3-phenylcoumarin, a
2-phenyl-4-quinoline, a 1-triflavone, a thioflavin, a benzoic acid
derivative, a naturally occurring alkaloid, a steroid and a
non-steriod anti-inflammatory compound (NSAID) may be used to
stimulate thiol-containing compound transport. For use within the
context of the embodiments they have the ability to stimulate
thiol-containing compound transport in tissues (i.e. epitheilial
tissues). The ability to stimulate thiol-containing compound
transport may be assessed using any of a variety of systems. For
example, in vitro assays using an epithelial cell line such as
human lung epithelial A549 (CFTR+) cells or rat lung epithelial
RL65 cells (CFTR+), human pancreas epithelial BxPC-3 (CFTR-) or
HTB-79 (CFTR+) cells, human colorectal epithelial HT-29 (CFTR+)
cells may be treated with at least one of the above compounds and
the level of thiol-containing compound transport measured.
[0187] Alternatively, the ability to stimulate thiol-containing
compound transport may be evaluated within an in vivo assay
employing a rodent species that has been genetically engineered to
either overexpress or under express apical GSH transporters (i.e.
CFTR, MDR or MRP). In general, such assays employ cell monolayers,
which may be prepared by standard cell culture techniques.
Alternatively, thiol-containing compound transport may be evaluated
using epithelial tissue in which the thiol-containing compound
across the apical membrane. In either system, thiol-containing
compound transportation is evaluated in the presence and absence of
a test compound (i.e., a flavone or isoflavone etc.), and those
compounds that stimulate thiol-containing compound transport as
described above may be used within the methods provided herein.
[0188] In one embodiment, dexamethasone may be used as a
therapeutic to stimulate thiol-containing compound transport.
Dexamethasone was chosen out of a long potential list of inducers
based on its ability to induce at least two ABC transporters: MRP-2
and MDR-1. This maximizes the chances of raising ELF GSH levels
through 2 separate pathways.
[0189] Other suitable therapeutic compounds may be identified using
the representative assays as described herein.
[0190] Flavones and isoflavones may generally be prepared using
well known techniques, such as those described (Shakhova et al.,
Zh. Obshch. Khim. 32:390, 1962; Farooq et al., Arch. Pharm.
292:792, 1959; and Ichikawa et al., Org. Prep. Prog. Int. 14:183,
1981). Alternatively, such compounds may be commercially available
(e.g., from Indofine Chemical Co., Inc., Somerville, N.J. or
Sigma-Aldrich, St. Louis, Mo.). Further modifications to such
compounds may be made using conventional organic chemistry
techniques, which are well known to those of ordinary skill in the
art. Most of the compound examples have published methods for
synthesis and referenced in the Merck Index (ed. 13.sup.th,
2001).
Nucleic Acids
[0191] As described herein, an aspect of the present disclosure
concerns isolated nucleic acids and methods of use of isolated
nucleic acids. The term "nucleic acid" is intended to include DNA
and RNA and can be either be double-stranded or single-stranded. In
a preferred embodiment, the nucleic acid is a cDNA comprising a
nucleotide sequence such as found in GenBank (i.e. human MDR-1 Gen
Bank accession #M2943). In certain embodiments, the nucleic acid
sequences disclosed herein have utility as hybridization probes or
amplification primers. These nucleic acids may be used, for
example, in diagnostic evaluation of tissue samples. In certain
embodiments, these probes and primers consist of oligonucleotide
fragments. Such fragments should be of sufficient length to provide
specific hybridization to a RNA or DNA tissue sample. The sequences
typically will be 10-20 nucleotides, but may be longer. Longer
sequences greater than 50 even up to full length, are preferred for
certain embodiments.
[0192] Accordingly, the nucleotide sequences may be used for their
ability to selectively form duplex molecules with complementary
stretches of genes or RNAs or to provide primers for amplification
of DNA or RNA from tissues. Those that are skilled in the art know
the stringency needed for effective hybridization of the
complementary component.
[0193] Many ABC transporters have been cloned (i.e. MDR1, MRP1,
MRP2 have been sequenced in their entirety). Embodiments of this
invention include induction of thiol-containing compound
transporter proteins using gene fusion (i.e. MRP:lacZ for MRP1 or
MDR expression) technologies known to those skilled in the art.
Other embodiments include the induction of thiol-containing
compound transporter genes via stimulation by a factor that binds
and or is known to stimulate the synthesis of the sequence of
interest (i.e. SP-1) by introducing said factor to a cell or
tissue. Other embodiments include the transport of the
thiol-containing compound transporter genes via a vesicle or
liposome for subsequent expression in the cell or tissue of
interest (i.e. lung epithelial cells, liver, pancreas,
gastrointestinal cells).
[0194] The following codon chart may be used to produce nucleic
acids encoding the same or slightly different amino acid sequences
of a given nucleic acid:
TABLE-US-00001 TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG
GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic
acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA
GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
[0195] In certain embodiments, it will be advantageous to employ
nucleic acid sequences in combination with an appropriate means,
such as a label, for determining hybridization. A wide variety of
appropriate indicator means are available (i.e. fluorcscent,
radioactive, enzymatic or other ligands, such as avidin/biotin)
that are capable of being detected. In preferred embodiments, one
may desire to employ a fluorescent label or an enzyme tag such as
urease, alkaline phosphatase or peroxidase, instead of radioactive
or other environmentally undesirable reagents. In the case of
enzyme tags, colorimetric indicator substrates are known which can
be employed to provide a detection means visible to the human eye
or spectrophotometrically, to identify specific hybridization with
complementary nucleic acid-containing samples.
[0196] In general, it is envisioned that the hybridization probes
described herein will not only be useful in solutions as in PCR,
for detection of expression of corresponding genes (i.e.
thiol-containing compound transporter) but also in embodiments
employing a solid phase. In embodiments involving a solid phase,
the test DNA (or RNA) is adsorbed or otherwise affixed to a
selected matrix or surface. This fixed, single-stranded nucleic
acid is then subjected to hybridization with selected probes under
known conditions.
[0197] The gene or gene fragment encoding a polypeptide (i.e. a
thiol-containing compound transporter) may be inserted into an
expression vector by standard subcloning techniques. An E. coli
expression vector may be used which produces the recombinant
polypeptide as a fusion protein, allowing rapid affinity
purification of the protein. Examples of such fusion protein
expression systems are the FLAG system (IBI, New Haven, Conn.), and
the 6.times.His system (Qiagen, Chatsworth, Calif.).
[0198] Inducible non-fusion expression vectors include pTrc (Amann
et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990) 60-89). While target gene expression
relies on host RNA polymerase transcription from the hybrid trp-lac
fusion promoter in pTrc, expression of target genes inserted into
pET 11d relies on transcription from the T7 gn10-lac 0 fusion
promoter mediated by coexpressed viral RNA polymerase (T7 gnl).
This viral polymerase is supplied by host strains BL21 (DE3) or
HMS174(DE3) from a resident lambda prophage harboring a T7 gnl
under the transcriptional control of the lacUV 5 promoter.
[0199] Examples of vectors for expression in yeast S. cerivisae
include pYepSec1 (Baldari. et al., (1987) Embo J. 6:229-234), pMFa
(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et
al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation,
San Diego, Calif.).
[0200] Baculovirus vectors available for expression of proteins in
cultured insect cells (SF 9 cells) include the pAc series (Smith et
al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series
(Lucklow, V. A., and Summers, M. D., (1989) Virology
170:31-39).
[0201] Expression of a thiol-containing compound transporter
protein in mammalian cells may be accomplished using a mammalian
expression vector. Examples of mammalian expression vectors include
pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al.
(1987), EMBO J. 6:187-195). The expression vector's control
functions are often provided by viral material (i.e. polyoma,
Adenovirus 2, cytomegalovirus and often, Simian Virus 40). The
pRc/CMV vector, nucleic acid introduced into the vector to be
expressed is under the control of the enhancer/promoter sequence
from the immediate early gene of human cytomegalovirus.
Additionally, the vector encodes a gene conferring neomycin
resistance. In one embodiment, the recombinant expression vector is
capable of directing expression of the nucleic acid preferentially
in a particular cell type. This means that the expression vector's
control functions are provided by regulatory sequences which allow
for preferential expression of a nucleic acid contained in the
vector in a particular cell type, thereby allowing for tissue or
cell-type specific expression of an encoded protein. For example, a
nucleic acid encoding a protein with thiol-containing compound
transporter activity can be preferentially expressed in lung cells
using promoter and enhancer sequences from a gene which is
expressed preferentially in epithelial cell lines such as human
lung epithelial A549 (CFTR+) cells or rat lung epithelial RL65
cells (CFTR+), human pancreas epithelial BxPC-3 (CFTR-) or HTB-79
(CFTR+) cells, and human colorectal epithelial HT-29 (CFTR+)
cells.
[0202] The recombinant expression vector may be a plasmid. The
recombinant expression vector further may be a virus, or portion
thereof, which allows for expression of a nucleic acid introduced
into the viral nucleic acid. For example, replication defective
retroviruses, adenoviruses and adeno-associated viruses can be
used.
[0203] Plasmid vectors introduced into mammalian cells are
integrated into host cell DNA at only a low frequency. In order to
identify these integrants, a gene that contains a selectable marker
(i.e., resistance to antibiotics) is generally introduced into the
host cells along with the gene of interest. Preferred selectable
markers include those that confer resistance to certain drugs, such
as G418 and hygromycin. Selectable markers can be introduced on a
separate plasmid from the nucleic acid of interest or, preferably,
are introduced on the same plasmid. Host cells transformed with one
or more recombinant expression vectors containing a nucleic acid
and a selectable marker may be identified by locating the marker.
For example, if the selectable marker encoded a gene conferring
neomycin resistance (such as pRc/CMV), transformant cells can be
selected with G418. Cells that have incorporated the selectable
marker genc will survive, while the other cells die.
[0204] Alternatively, the protein or parts thereof can be prepared
by chemical synthesis using techniques well known in the chemistry
of proteins such as solid phase synthesis (Merrifield, 1964, J. Am.
Chem. Assoc. 85:2149-2154) or synthesis in homogeneous solution
(Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch,
Vol. 15 I and II, Thieme, Stuttgart).
[0205] For applications in which the nucleic acid segments are
incorporated into vectors, such as plasmids, cosmids or viruses,
these segments may be combined with other DNA sequences, such as
promoters, polyadenylation signals, restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably.
[0206] Promoters that are most commonly used in recombinant DNA
construction include the .beta.-lactamase (penicillinase), lactose
and tryptophan (trp) promoter systems. While these are the most
commonly used, other microbial promoters have been discovered and
utilized, and details concerning their nucleotide sequences have
been published, enabling those of skill in the art to ligate them
functionally with plasmid vectors.
[0207] Suitable promoting sequences in yeast vectors include the
promoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980) or
other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978),
such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. In constructing suitable expression plasmids, the
termination sequences associated with these genes are also ligated
into the expression vector 3' of the sequence desired to be
expressed to provide polyadenylation of the mRNA and
termination.
[0208] Other suitable promoters, which have the additional
advantage of transcription controlled by growth conditions, include
the promoter region for alcohol dehydrogenase 2, isocytochrome C,
acid phosphatase, degradative enzymes associated with nitrogen
metabolism, and the aforementioned glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization
[0209] The recombinant expression vectors can be designed for
expression of thiol-containing compound transporter proteins in
prokaryotic or eukaryotic cells. For example, proteins can be
expressed in bacterial cells such as E. coli, insect cells (using
baculovirus), yeast cells or mammalian cells.
[0210] Expression in prokaryotes is most often carried out in E.
coli with vectors containing constitutive or inducible promotors
directing the expression of either fusion or non-fusion proteins.
Fusion vectors add a number of amino acids usually to the amino
terminus of the expressed target gene. Such fusion vectors
typically serve three purposes: 1) to increase expression of
recombinant protein; 2) to increase the solubility of the target
recombinant protein; and 3) to aid in the purification of the
target recombinant protein by acting as a ligand in affinity
purification. Often, in fusion expression vectors, a proteolytic
cleavage site is introduced at the junction of the fusion moiety
and the target recombinant protein to enable separation of the
target recombinant protein subsequent to purification of the fusion
protein (i.e. enzymes, and their cognate recognition sequences,
such as Factor Xa, thrombin and enterokinase). Typical fusion
expression vectors include pGEX (Amrad Corp., Melbourne,
Australia), pMAL (New England Biolabs, Beverly, Md.) and pRIT5
(Pharmacia, Piscataway, N.J.) that fuse maltose E binding protein,
or protein A, respectively, to the target recombinant protein.
[0211] DNA segments encoding a specific thiol-containing compound
transporter gene may be introduced into recombinant host cells and
employed for expressing a specific structural or regulatory
protein. Alternatively, through the application of genetic
engineering techniques, subportions or derivatives of selected
genes may be employed.
[0212] Where an expression product is to be generated, it is
possible for the nucleic acid sequence to be varied while retaining
the ability to encode the same product. Reference to the codon
chart, provided above, will permit those of skill in the art to
design any nucleic acid encoding for the product of a given nucleic
acid.
[0213] One embodiment includes isolated nucleic acids encoding
proteins having biological activity of thiol-containing compound
transporters. The term "isolated" refers to a nucleic acid
substantially free of cellular material or culture medium when
produced by recombinant DNA techniques, or chemical precursors or
other chemicals when chemically synthesized. An "isolated" nucleic
acid is also free of sequences that naturally flank the nucleic
acid (i.e., sequences located at the 5' and 3' ends of the nucleic
acid) in the organism from which the nucleic acid is derived.
[0214] It will be appreciated that isolated nucleic acids includes
nucleic acids having substantial sequence homology with the
nucleotide sequence of the thiol-containing compound transporter
found in GenBank as disclosed in methods found herein or encoding
proteins having substantial homology to the corresponding amino
acid sequence.
[0215] Some MRP sequences are highly conserved (i.e. MRP-2). There
are 12 hydrophobic stretches predicted to be membrane-spanning
regions and of functional importance. These regions are important
to maintain the integrity of the transporter (U.S. Pat. No.
5,766,880). In addition there are two regions having the structural
characteristics of nucleotide binding folds (NBFs) typical of
ATP-binding cassette domains (ABC domains). See Hyde, S. C. et al.,
Nature 346, 362-365 (1990). Part of the structure of these NBFs are
conserved in other members of the ABC superfamily of membrane
transport proteins. They bind nucleotides and are functionally
important. See Higgins, C. F., Ann. Rev. Cell Biol. 8, 67-113
(1992). These regions must be conserved for functional activity.
Alternatively, nucleotide and corresponding amino acid
substitutions that maintain the structure of an NBF are likely to
be tolerated: In addition, some nucleotides encoding an NBF of one
member of the ABC superfamily of membrane transport proteins can be
substituted for the homologous domain of another member while
maintaining function of the protein. (Buschman, F. and Gros, P.
Mol. Cell. Biol. 11, 595-603 (1991).
[0216] Proteins comprising an amino acid sequence that is 50%, 60%,
70%, 80% or 90% homologous with the amino acid may provide proteins
having thiol-containing compound transporter activity. The
embodiments encompass a nucleic acid encoding a protein having
biological activity of a thiol-containing compound transporter
which is at least 25% homologous with the amino acid sequence
discussed previously and other yet unknown thiol-containing
compound transporters (Borst BBA 1461:347 (1999)).
[0217] It will further be appreciated that variant forms of the
nucleic acids that arise by alternative splicing of an mRNA
corresponding to a cDNA are encompassed by the methods.
[0218] Isolated nucleic acids encoding a protein having the
biological activity of a thiol-containing compound transporter, as
described herein, and having a sequence that differs from a
nucleotide sequence due to degeneracy in the genetic code are also
within the scope. As one example, DNA sequence polymorphisms within
the nucleotide sequence of a thiol-containing compound transporter
protein (especially those within the third base of a codon) may
result in "silent" mutations in the DNA that do not affect the
amino acid encoded. However, it is expected that DNA sequence
polymorphisms that do lead to changes in the amino acid sequences
of a thiol-containing compound transporter protein will exist
within a population. Any and all such nucleotide variations and
resulting amino acid polymorphisms are within the scope.
Furthermore, there may be one or more isoforms or related,
cross-reacting family members of the thiol-containing compound
transporter(s) described herein. Such isoforms or family members
are defined as proteins related in biological activity and amino
acid sequence to thiol-containing compound transporter, but encoded
by genes at different loci.
[0219] Since thiol-containing compounds are often co-transported
with certain drugs, an isolated nucleic acid encoding a protein
having the biological activity of thiol-containing compound
transporter can be isolated in certain situations from a multidrug
resistant cell line which displays a predetermined level of
resistance to such drugs as anthracyclines, epipodophyllotoxins and
Vinca alkaloids. One example of such a cell line is the H69AR
described above. Other suitable cell lines can be produced by
stepwise selection of a resistant cell lines in the presence of
increasing concentrations of a drug for which resistance is to be
acquired over a period of several months to years. A multi-drug
resistance cell line is evaluated by exposing it to other drug(s)
(i.e vincristine) and determining the cytotoxicity of that drug for
the cell line. Once a cell line is identified, a nucleic acid is
isolated by preparing a cDNA library from this cell line by
standard techniques and screening this library with cDNA produced
from total mRNA isolated from the cell line and its drug sensitive
parental cell line (i.e. H69AR vs. H69 cells). The library is
plated and replica filters are prepared by standard methods. Each
set of filters is screened with cDNA prepared from the respective
mRNA (i.e. experimental vs. parental). Those cDNA clones displaying
increased hybridization with the experimental cDNA when compared to
the parental cDNA can be selected from the library. For
descriptions of differential cDNA library screening see King, C.
R., et al. J. Biol. Chem. 254, 6781 (1979); Van der Bliek, A. M.,
et al., Mol. Cell. Biol. 6, 1671 (1986).
[0220] Determination of whether a cDNA so isolated has the
biological activity of a thiol-containing compound transporter can
be accomplished by expressing the cDNA in a parental mammalian
cell, by standard techniques, and assessing whether expression in
the cell of the protein encoded by the cDNA confers on the cell the
ability to transport thiol-containing compounds used in its
isolation and identification. A cDNA having the biological activity
of a thiol-containing compound transporter so isolated may be
sequenced by standard techniques, such as dideoxynucleotide chain
termination or Maxam-Gilbert chemical sequencing, to determine the
nucleic acid sequence and the predicted amino acid sequence of the
encoded protein.
[0221] Alternatively, a genomic DNA library can be similarly
screened to isolate a genomic clone encompassing a gene encoding a
protein having thiol-containing compound transporter activity. A
human thiol-containing compound transporter gene has been
previously mapped to chromosome 16 (MRP, U.S. Pat. No. 5,766,880).
Therefore, a chromosome 16 library rather than a total genomic DNA
library can also be used to isolate a human thiol-conatining
transporter gene(s). Nucleic acids isolated by screening of a cDNA
or genomic DNA library can be sequenced by standard techniques.
[0222] An isolated nucleic acid that is DNA can also be isolated by
selectively amplifying a nucleic acid encoding a protein having
thiol-containing compound transporter activity using the polymerase
chain reaction (PCR) method and genomic DNA or mRNA. cDNA from mRNA
can be prepared by a variety of well-known techniques (i.e. by
using the guanidinium-thiocyanate extraction procedure of Chirgwin
et al., Biochemistry, 18, 5294-5299 (1979).) It is possible to
design synthetic oligonucleotide primers from the nucleotide
sequence for use in a PCR reaction. A nucleic, acid can be
amplified from cDNA or genomic DNA using these oligonucleotide
primers and standard PCR amplification techniques. The nucleic acid
so amplified can be cloned into an appropriate vector and
characterized by DNA sequence analysis.
[0223] An isolated nucleic acid of the embodiments that is RNA can
be isolated by cloning a cDNA into an appropriate vector which
allows for transcription of the cDNA to produce an RNA molecule
which encodes a protein having thiol-containing compound
transporter activity. For example, a cDNA can be cloned downstream
from a promoter (such as T7 and induced by T7 polymerase). The RNA
product can be isolated by standard techniques.
[0224] A nucleic acid of the embodiments, for instance an
oligonucleotide, can also be chemically synthesized using standard
techniques. Various methods of chemically synthesizing
polydeoxynucleotides are known, including solid-phase synthesis
which, like peptide synthesis, has been fully automated in
commercially available DNA synthesizers (See i.e., Itakura et al.
U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066;
and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071).
[0225] The identification of the initiation codon and untranslated
sequences of a thiol-containing compound transporter can be
evaluated using currently available computer software designed for
the purpose (i.e. PC/Gene--IntelliGenetics Inc., Calif.). The
intron/exon structure and the transcription regulatory sequences of
the gene encoding the thiol-containing compound transporter cDNA
can be identified using a nucleic acid to probe a genomic DNA clone
library. Regulatory elements, such as promoter and enhancers
necessary for expression of the gene encoding the thiol-containing
compound transporter in various tissues, can be identified using
conventional techniques. The function of the elements can be
confirmed by using them to express a reporter gene such as the
bacterial gene lacZ that is operatively linked to the fragments.
Such a construct can be introduced into cultured cells using
standard procedures or into non-human transgenic animal models. In
addition to identifying regulatory elements in DNA, such constructs
can also be used to identify nuclear proteins interacting with said
elements, using techniques known in the art.
[0226] The isolated nucleic acids or oligonucleotide fragments of
the isolated nucleic acids allow construction of nucleotide probes
for use in the detection of nucleotide sequences in biological
materials, such CF patient lung cells. A nucleotide probe can be
labelled with a radioactive element which provides for an adequate
signal as a means for detection and has sufficient half-life to be
useful for detection, such as .sup.32P, .sup.3H, .sup.14C or the
like. Other materials that can be used to label the probe include
antigens that are recognized by a specific labelled antibody,
fluorescent compounds, enzymes, antibodies specific for a labelled
antigen, and chemiluminescent compounds.
[0227] The nucleic acids can confer increases in thiol-containing
compound transport due to exposure to drugs such as anthracyclines,
cis platinum, bleomycin, epipodophyllotoxins and Vinca alkaloids on
a drug sensitive cell when transfected into the cell. As well as
conferring increased transport of thiol-containing compounds, these
drugs can serve as selecting agents when preparing a transformant
host cell rather than using an independent selectable marker (such
as neomycin resistance). (Croop et al., U.S. Pat. No. 5,198,344).
Cells may be selected by exposure to one or more drugs for which
thiol-containingcompound transport increase is conferred by the
nucleic acid expression.
[0228] An isolated nucleic acid can be tested for thiol-containing
compound transporter activity by incorporating the nucleic acid
into a recombinant expression vector, transforming a mammalian cell
with the recombinant expression vector to make a transformant host
cell as described above and testing the ability to excrete
thiol-containing compound(s). For example, in a preferred
embodiment, the transformant host cell is an epithelial cell, and
the thiol-containing compound transporter ability of transfected
epithelial cell is compared to that of untransfected epithelial
cell or preferably to epithelial cells transfected with the
parental expression vector lacking the nucleic acid encoding a
protein having thiol-containing compound transporter activity. One
embodiment includes the increase in thiol-containing compound
transporter activity. Other embodiments include increased apical
localized thiol-containing compound transporter activity.
Protein Purification
[0229] Various methods for quantifying the degree of purification
of the protein or peptide will be known to those of skill in the
art in light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
analysis by SDS/PAGE to identify the number of polypeptides in a
given fraction. A preferred method for assessing the purity of a
fraction is to calculate the specific activity of the fraction, to
compare it to the specific activity of the initial extract, and to
thus calculate the degree of purity, herein assessed by a "-fold
purification number". The actual units used to represent the amount
of activity will be dependent upon the particular assay technique
chosen to follow the purification and whether or not the expressed
protein or peptide exhibits a detectable activity.
[0230] Methods for purifying various forms of proteins are known.
(i.e., Protein Purification, ed. Scopes, Springer-Verlag, New York,
N.Y., 1987; Methods in Molecular Biology: Protein Purification
Protocols, Vol. 59, ed. Doonan, Humana Press, Totowa, N.J., 1996).
The methods disclosed in the cited references are exemplary only
and any variation known in the art may be used. Where a protein is
to be purified, various techniques may be combined, including but
not limited to cell fractionation, column chromatography (e.g.,
size exclusion, ion exchange, reverse phase, affinity, etc.), Fast
Performance Liquid Chromatography (FPLC), High Performance Liquid
Chromatography (HPLC), gel electrophoresis, precipitation with
salts, pH, organic solvents or antibodies, ultrafiltration and/or
ultracentrifugation.
[0231] There is no general requirement that the protein or peptide
always be provided in the most purified state. Indeed, it is
contemplated that less substantially purified products will have
utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or
by utilizing different forms of the same general purification
scheme. Methods exhibiting a lower degree of relative purification
may have advantages in total recovery of protein product, or in
maintaining the activity of an expressed protein.
[0232] One embodiment provides isolated proteins having biological
activity of a thiol-containing compound transporter (i.e. MRP2,
MDR1 etc.). In a preferred embodiment the protein having biological
activity of a thiol-containing compound transporter comprises an
amino acid sequence found in GenBank (i.e. human p-glycoprotein,
MDR-1 GenBank accession #M29432). Proteins having biological
activity of a thiol-containing compound transporter that have
substantial sequence homology to the amino acid sequence of an ABC
transporter as defined above, are also encompassed herein.
Furthermore, proteins having biological activity of a
thiol-containing compound transporter that are encoded by nucleic
acids which hybridize under high or low stringency conditions to a
nucleic acid comprising a nucleotide sequence described previously
are encompassed. Preferred immunogenic portions correspond to
regions of the protein not conserved in other ABC superfamily
members, (i.e. outside of the two NBF domains), and include regions
between the 12 membrane spanning regions. An immunogenic portion
will be of at least about eight amino acids in length.
[0233] Molecules which bind to a protein including the antibodies,
bispecific antibodies and tetrameric antibody complexes, can be
used in a method for identifying thiol-containing compound
transporters by labelling the molecule with a detectable substance,
contacting the molecule with cells and detecting the detectable
substance bound to the cells. A molecule which binds to a protein
may be used in a method for increasing the activity of the
thiol-containing compound transporter (i.e. by inhibiting the
secretion of interfering compounds and/or activating the excretion
of thiol-containing compound secretion).
Antibodies
[0234] The proteins used in the methods, or portions thereof, can
be used to prepare antibodies specific for the proteins. Antibodies
can be prepared which bind a distinct epitope in an unconserved
region of the protein. An unconserved region of the protein is one
that does not have substantial sequence homology to other proteins,
for example other members of the ABC superfamily of membrane
transport proteins. For example, unconserved regions encompassing
sequences between the twelve membrane spanning regions mentioned
previously and excluding conserved regions (i.e. the NBF domains),
can be used. Alternatively, a region from one of the two NBF
domains can be used to prepare an antibody to a conserved region of
a thiol-containing compound transporter protein. An antibody to a
conserved region may be capable of reacting with other members of
the ABC family of membrane transport proteins. Conventional methods
can be used to prepare the antibodies. For example, by using a
peptide of a thiol-containing compound transporter protein,
polyclonal antisera or monoclonal antibodies can be made using
standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can
be immunized with an immunogenic form of the peptide that elicits
an antibody response in the mammal. Techniques for conferring
immunogenicity on a peptide include conjugation to carriers or
other techniques using kits are well known in the art (Pierce
Biochemical). For example, the peptide can be administered in the
presence of adjuvant. The progress of immunization can be monitored
by detection of antibody titers in plasma or serum. Standard ELISA
or other immunoassay can be used with the immunogen as antigen to
assess the levels of antibodies. Following immunization, antisera
can be obtained and, if desired, polyclonal antibodies isolated
from the sera. One embodiment includes the use of specific
antibodies that enhance the transport of a thiol-containing
compound via a thiol-containing compound transporter possibly by
inhibiting a molecule that otherwise decreases the transporter
activity. Other embodiments include the use of specific antibodies
to inhibit the activity of factors that inhibit thiol-containing
compound transport activity.
[0235] To produce monoclonal antibodies, techniques are well known
in the art. For example, the hybridoma technique originally
developed by Kohler and Milstein (Nature 256, 495-497 (1975)) as
well as other techniques such as the human B-cell hybridoma
technique (Kozbor et al., Immunol. Today 4, 72 (1983)). Hybridoma
cells can be screened immunochemically for production of antibodies
specifically reactive with the peptide and monoclonal antibodies
isolated.
[0236] When antibodies produced in non-human subjects are used
therapeutically in humans, they are recognized to varying degrees
as foreign and an immune response may be generated in the patient.
One approach for minimizing or eliminating this problem, which is
preferable to general immunosuppression, is to produce chimeric
antibody derivatives, i.e., antibody molecules that combine a
non-human animal variable region and a human constant region.
Chimeric antibody molecules can include, for example, the antigen
binding domain from an antibody of a mouse, rat, or other species,
with human constant regions. A variety of approaches for making
chimeric antibodies have been described and may be used to make
chimeric antibodies containing the immunoglobulin variable region
that recognizes the gene product of the thiol-containing compound
transporter genes. See, for example, Morrison et al., Proc. Natl.
Acad. Sci. U.S.A. 81, 6851 (1985); Takeda et al., Nature 314, 452
(1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S.
Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication
EP171496; European Patent Publication 0173494, United Kingdom
Patent GB 2177096B. It is expected that such chimeric antibodies
would be less immunogenic in a human subject than the corresponding
non-chimeric antibody.
[0237] For human therapeutic purposes the monoclonal or chimeric
antibodies specifically reactive with a protein, or peptide
thereof, having the biological activity of a thiol-containing
compound transporter as described herein can be further humanized
by producing human constant region chimeras, in which parts of the
variable regions, especially the conserved framework regions of the
antigen-binding domain, are of human origin and only the
hypervariable regions are of non-human origin. Such altered
immunoglobulin molecules may be made by any of several techniques
known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci.
U.S.A., 80, 7308-7312 (1983).
[0238] Another method of generating specific antibodies, or
antibody fragments, reactive against protein, or peptide thereof,
having the biological activity of a thiol-containing compound
transporter is to screen expression libraries encoding
immunoglobulin genes, or portions thereof, expressed in bacteria
with peptides produced from the nucleic acid molecules. (Ward et
al., Nature 341, 544-546: (1989); Huse et al., Science 246,
1275-1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990).
Screening such libraries with, for example, a thiol-containing
compound transporter peptide can identify immunoglobulin fragments
reactive with a thiol-containing compound transporter.
[0239] The polyclonal, monoclonal or chimeric monoclonal antibodies
can be used to detect the proteins of the methods, portions thereof
or closely related isoforms in various biological materials, for
example they can be used in a radioimmunoassay, histochemical or in
an Elisa test. Thus, the antibodies can be used to quantify the
amount of a thiol-containing compound transporter protein of the
methods. The antibodies of the methods can be used to determine the
role of a thiol-containing compound transporter protein in cellular
events, particularly its role in thiol-containing compound
transport.
[0240] The polyclonal or monoclonal antibodies can be coupled to a
detectable substance such as enzymes (i.e. horseradish peroxidase,
alkaline phosphatase, glucose oxidase and galactosidase) and
luminescent material such as luminol; and radioactive material such
as .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0241] The embodiments provide a method for identifying a
thiol-containing compound transporter(s) using the disclosed
activating agents, proteins, nucleic acids and antibodies. One
embodiment further provides methods for increasing the
thiol-containing compound transporter activity and/or expression.
Furthermore, another embodiment provides diagnostic kits for
identifying thiol-containing compound transporters.
[0242] The compositions are administered to subjects in a
biologically compatible form suitable for pharmaceutical
administration in vivo. By "biologically compatible form suitable
for administration in vivo" is meant a form of the active agent
(i.e. pharmaceutical chemical, protein, gene, antibody etc of the
embodiments) to be administered in which any toxic effects are
outweighed by the therapeutic effects of the active agent.
Administration of a therapeutically active amount of the
therapeutic compositions is defined as an amount effective, at
dosages and for periods of time necessary to achieve the desired
result. For example, a therapeutically active amount of an antibody
reactive with a thiol-containing compound transporter protein may
vary according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of antibody to elicit a
desired response in the individual. Dosage regima may be adjusted
to provide the optimum therapeutic response. For example, several
divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation.
[0243] In one embodiment, the compound (i.e. pharmaceutical
chemical, gene, protein, antibody etc of the embodiments) may be
administered in a convenient manner such as by injection such as
subcutaneous, intravenous, by oral administration, inhalation,
transdermal application, intravaginal application, topical
application, intranasal or rectal administration. Depending on the
route of administration, the active compound may be coated in a
material to protect the compound from the degradation by enzymes,
acids and other natural conditions that may inactivate the
compound. In a preferred embodiment, the compound may be orally
administered. In another preferred embodiment, the compound may be
inhaled in order to make the compound bioavailable to the lung.
[0244] A compound may be administered to a subject in an
appropriate carrier or diluent, co-administered with enzyme
inhibitors or in an appropriate carrier such as liposomes. The term
"pharmaceutically acceptable carrier" as used herein is intended to
include diluents such as saline and aqueous buffer solutions. To
administer a compound that stimulates a thiol-containing compound
transporter protein by other than parenteral administration, it may
be necessary to coat the compound with, or co-administer the
compound with, a material to prevent its inactivation. Enzyme
inhibitors include pancreatic trypsin inhibitor,
diisopropylfluorophosphate (DEP) and trasylol. Liposomes include
water-in-oil-in-water emulsions as well as conventional liposomes
(Strejan et al., (1984) J. Neuroimmunol 7:27). The active agent may
also be administered parenterally or intraperitoneally. Dispersions
can also be prepared in glycerol, liquid polyethylene glycols, and
mixtures thereof and in oils. Under ordinary conditions of storage
and use, these preparations may contain a preservative to prevent
the growth of microorganisms.
[0245] 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. In all cases, the
composition must be sterile and must 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 pharmaceutically acceptable carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyetheylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of microorganisms can be achieved by various antibacterial and
antifungal agents (i.e., 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. A compound such as aluminum monostearate and gelatin
can be included to prolong absorption of the injectable
compositions.
[0246] Sterile injectable solutions can be prepared by
incorporating active compound (i.e. a chemical that increases the
activity of thiol-containing compound transporter protein) 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
dispersion medium and other required ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient (i.e. a chemical agent, antibody
etc.) plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0247] When the active agent is suitably protected, as described
above, the composition may be orally administered (or otherwise
indicated), for example, with an inert diluent or an assimilable
edible carrier. It is especially advantageous to formulate
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms are dictated by and directly dependent on (a) the unique
characteristics of the active agent and the particular therapeutic
effect to be achieved, and (b) the limitations inherent an active
agent for the therapeutic treatment of individuals.
[0248] Aqueous compositions comprise an effective amount of a
therapeutic protein, compound, peptide, epitopic core region,
stimulator (i.e. dexamethasone, rutin, MDR-2 protein), inhibitor,
and the like, dissolved or dispersed in a pharmaceutically
acceptable carrier or aqueous medium. Aqueous compositions of gene
therapy vectors expressing. any of the foregoing are also
contemplated.
[0249] Aqueous compositions comprise an effective amount of the
compound, dissolved or dispersed in a pharmaceutically acceptable
carrier or aqueous medium. Such compositions can also be referred
to as inocula. As used herein, "pharmaceutically acceptable
carrier" includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions. For human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologics
standards.
[0250] The biological material should be extensively dialyzed to
remove undesired small molecular weight molecules and/or
lyophilized for more ready formulation into a desired vehicle,
where appropriate. The active compounds will then generally be
formulated for parenteral administration (i.e. formulated for
injection via the intravenous, intramuscular, sub-cutaneous,
intralesional, or even intraperitoneal routes). The preparation of
an aqueous composition that contains an active component or
ingredient will be known. Typically, such compositions can be
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for use in preparing solutions or suspensions
upon the addition of a liquid prior to injection can also be
prepared; and the preparations can also be emulsified.
[0251] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid. 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.
[0252] Solutions of the active compounds as free-base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0253] A therapeutic agent can be formulated into a composition in
a neutral or salt form. Pharmaceutically acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. In terms of using
peptide therapeutics as active ingredients, the technology of U.S.
Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;
and 4,578,770, each incorporated herein by reference, may be
used.
[0254] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
preparation of more, or highly, concentrated solutions for direct
injection is also contemplated, where the use of DMSO as solvent is
envisioned to result in extremely rapid penetration, delivering
high concentrations of the active agents to a small area.
[0255] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0256] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media that can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and
1570-1580).
[0257] The active therapeutic agents may be formulated within a
mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001
to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams
per dose or so. Multiple doses can also be administered.
[0258] In addition to the compounds formulated for parenteral
administration, such as intravenous or intramuscular injection,
other pharmaceutically acceptable forms include, e.g., tablets or
other solids for oral administration; liposomal formulations;
time-release capsules; and any other form currently used.
[0259] In another embodiment, nasal solutions or sprays, aerosols
or inhalants may be used to deliver the compound of interest. Nasal
solutions are usually aqueous solutions designed to be administered
to the nasal passages in drops or sprays. Nasal solutions are
prepared so that they are similar in many respects to nasal
secretions. Thus, the aqueous nasal solutions usually are isotonic
and slightly buffered to maintain a pH of 5.5 to 6.5. In addition,
antimicrobial preservatives, similar to those used in ophthalmic
preparations, and appropriate drug stabilizers, if required, may be
included in the formulation. Various commercial nasal preparations
are known and include, for example, antibiotics and antihistamines
and are used for asthma prophylaxis. Inhalation preparations may
include solutioins or dry powder formulations that are commonly
used along with a propellant in the formulation of therapeutics
used for the treatment of asthmatics.
[0260] Additional formulations that are suitable for other modes of
administration include suppositories and pessaries. A rectal
pessary or suppository may also be used. In general, for
suppositories, traditional binders and carriers may include, for
example, polyalkylene glycols or triglycerides; such suppositories
may be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, preferably 1%-2%.
[0261] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders. In certain defined embodiments, oral
pharmaceutical compositions will comprise an inert diluent or
assimilable edible carrier, or they may be enclosed in hard or soft
shell gelatin capsule, or they may be compressed into tablets, or
they may be incorporated directly with the food of the diet. For
oral therapeutic administration, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
should contain at least 0.1% of active compound. The percentage of
the compositions and preparations may, of course, be varied and may
conveniently be between about 2 to about 75% of the weight of the
unit, or preferably between 25-60%. The amount of active compounds
in such therapeutically useful compositions is such that a suitable
dosage will be obtained.
[0262] A pharmaceutical composition may be prepared with carriers
that protect active ingredients against rapid elimination from the
body, such as time-release formulations or coatings. Such carriers
include controlled release formulations, such as, but not limited
to, microencapsulated delivery systems, and biodegradable,
biocompatible polymers, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid
and others are known.
[0263] Particularly preferred are methods in which the therapeutic
compound(s) are directly administered as a pressurized aerosol or
nebulized formulation to the patient's lungs via inhalation. Such
formulations may contain any of a variety of known aerosol
propellants useful for endopulmonary and/or intranasal inhalation
administration. In addition, water may be present, with or without
any of a variety of cosolvents, surfactants, stabilizers (e.g.,
antioxidants, chelating agents, inert gases and buffers).
[0264] Pharmaceutical compositions are administered in an amount,
and with a frequency, that is effective to inhibit or alleviate the
symptoms of a thiol-containing compound transporter deficient
condition (i.e. CF) and/or to delay the progression of the disease.
The precise dosage and duration of treatment may be determined
empirically using known testing protocols or by testing the
compositions in model systems known in the art and extrapolating
therefrom. Dosages may also vary with the severity of the disease.
A pharmaceutical composition is generally formulated and
administered to exert a therapeutically useful effect while
minimizing undesirable side effects. In general, an oral dose
ranges from about 200 mg to about 1000 mg, which may be
administered 1 to 3 times per day. It will be apparent that, for
any particular subject, specific dosage regimens may be adjusted
over time according to the individual need.
[0265] The tablets, troches, pills, capsules and the like may also
contain the following: a binder, as gum tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup of elixir may contain the active compounds
sucrose as a sweetening agent methyl and propylparabens as
preservatives, a dye and flavoring, such as grape or orange
flavor
[0266] In certain broad embodiments, the oligo- or polynucleotides
and/or expression vectors may be entrapped in a liposome. Liposomes
are vesicular structures characterized by a phospholipid bilayer
membrane and an inner aqueous medium. Multilamellar liposomes have
multiple lipid layers separated by aqueous medium. They form
spontaneously when phospholipids are suspended in an excess of
aqueous solution. The lipid components undergo self-rearrangement
before the formation of closed structures and entrap water and
dissolved solutes between the lipid bilayers (Ghosh and Bachhawat,
1991). Also contemplated are cationic lipid-nucleic acid complexes,
such as lipofectamine-nucleic acid complexes.
[0267] In certain embodiments, the liposome may be complexed with a
hemagglutinating virus (HVJ). This has been shown to facilitate
fusion with the cell membrane and promote cell entry of
liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the liposome may be complexed or employed in
conjunction with nuclear non-histone chromosomal proteins (HMG-1)
(Kato et al., 1991). In yet further embodiments, the liposome may
be complexed or employed in conjunction with both HVJ and HMG-1. In
that such expression vectors have been successfully employed in
transfer and expression of a polynucleotide in vitro and in vivo,
then they are applicable. Where a bacterial promoter is employed in
the DNA construct, it also will be desirable to include within the
liposome an appropriate bacterial polymerase.
[0268] Lipids suitable for use accordingly can be obtained from
commercial sources. For example, dimyristyl phosphatidylcholine
("DMPC") can be obtained from Sigma Chemical Co., dicctyl phosphate
("DCP") is obtained from K & K Laboratories (Plainview, N.Y.);
cholesterol ("Chol") is obtained from Calbiochem-Behring;
dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock
solutions of lipids in chloroform, chloroform/methanol or t-butanol
can be stored at about -20.degree. C. Preferably, chloroform is
used as the only solvent since it is more readily evaporated than
methanol.
[0269] Phospholipids from natural sources, such as egg or soybean
phosphatidylcholine, brain phosphatidic acid, brain or plant
phosphatidylinositol, heart cardiolipin and plant or bacterial
phosphatidylethanolamine are preferably not used as the primary
phosphatide, i.e., constituting 50% or more of the total
phosphatide composition, because of the instability and leakiness
of the resulting liposomes.
[0270] Liposomes used accordingly can be made by different methods.
The size of the liposomes varies depending on the method of
synthesis. A liposome suspended in an aqueous solution is generally
in the shape of a spherical vesicle, having one or more concentric
layers of lipid bilayer molecules. Each layer consists of a
parallel array of molecules represented by the formula XY, wherein
X is a hydrophilic moiety and Y is a hydrophobic moiety. In aqueous
suspension, the concentric layers are arranged such that the
hydrophilic moieties tend to remain in contact with an aqueous
phase and the hydrophobic regions tend to self-associate. For
example, when aqueous phases are present both within and without
the liposome, the lipid molecules will form a bilayer, known as a
lamella, of the arrangement XY-YX.
[0271] Liposomes within the scope can be prepared in accordance
with known laboratory techniques. In one preferred embodiment,
liposomes are prepared by mixing liposomal lipids, in a solvent in
a container, e.g., a glass, pear-shaped flask. The container should
have a volume ten-times greater than the volume of the expected
suspension of liposomes. Using a rotary evaporator, the solvent is
removed at approximately 40.degree. C. under negative pressure. The
solvent normally is removed within about 5 min to 2 hours,
depending on the desired volume of the liposomes. The composition
can be dried further in a desiccator under vacuum. The dried lipids
generally are discarded after about 1 week because of a tendency to
deteriorate with time.
[0272] The dried lipids or lyophilized liposomes prepared as
described above may be reconstituted in a solution of active agent
(i.e. nucleic acid, chemical agent, antibody etc.), and the
solution diluted to an appropriate concentration with a suitable
solvent known to those skilled in the art. The mixture is then
vigorously shaken in a vortex mixer. Unencapsulated active agent is
removed by centrifugation. The liposomes are washed resuspended at
an appropriate total phospholipid concentration, e.g., about 50-200
mM. The amount of active agent encapsulated can be determined in
accordance, with standard methods.
[0273] In a preferred embodiment, a nucleic acid (thiol-containing
compound transporter) and the lipid dioleoylphosphatidylcholine may
be employed. For example, nuclease-resistant oligonucleotides may
be mixed with lipids in the presence of excess t-butanol. The
mixture is vortexed before being frozen in an acetone/dry ice bath.
The frozen mixture is lyophilized and hydrated with Hepes-buffered
saline (1 mM Hepes, 10 mM NaCl, pH 7.5) overnight, and then the
liposomes are sonicated in a bath type sonicator for 10 to 15 min.
The size of the liposomal-oligonucleotides typically ranged between
200-300 nm in diameter as determined by the submicron particle
sizer autodilute model 370 (Nicomp, Santa Barbara, Calif.).
[0274] As a model system for eukaryotic gene expression,
adenoviruses have been widely studied and well characterized, which
makes them an attractive system for development of adenovirus as a
gene transfer system. This group of viruses is easy to grow and
manipulate, and they exhibit a broad host range in vitro and in
vivo. In lytically infected cells, adenoviruses are capable of
shutting off host protein synthesis, directing cellular machineries
to synthesize large quantities of viral proteins, and producing
copious amounts of virus.
[0275] The E1 region of the genome includes E1A and E1B that encode
proteins responsible for transcription regulation of the viral
genome, as well as a few cellular genes. E2 expression, including
E2A and E2B, allows synthesis of viral replicative functions (i.e.
DNA-binding protein, DNA polymerase, and a terminal protein that
primes replication). E3 gene products prevent cytolysis by
cytotoxic T cells and tumor necrosis factor and appear to be
important for viral propagation. Functions associated with the E4
proteins include DNA replication, late gene expression, and host
cell shutoff. The late gene products include most of the virion
capsid proteins, and these are expressed only after most of the
processing of a single primary transcript from the major late
promoter has occurred. The major late promoter (MLP) exhibits high
efficiency during the late phase of the infection
(Stratford-Perricaudet and Perricaudet, 1991).
[0276] Particular advantages of an adenovirus system for delivering
foreign proteins to a cell include (i) the ability to substitute
relatively large pieces of viral DNA by foreign DNA; (ii) the
structural stability of recombinant adenoviruses; (iii) the safety
of adenoviral administration to humans; and (iv) lack of any known
association of adenoviral infection with cancer or malignancies;
(v) the ability to obtain high titers of the recombinant virus; and
(vi) the high infectivity of adenovirus (vii) adenovirus
replication is independent of host gene replication, unlike
retroviral sequences and (viii) oncogenic risk from adenovirus
vectors is thought to be negligible (Grunhaus & Horwitz,
1992).
[0277] In general, adenovirus gene transfer systems are based upon
recombinant, engineered adenovirus that are rendered
replication-incompetent by deletion of a portion of its genome,
such as E1, and yet still retains its competency for infection.
Sequences encoding relatively large foreign proteins can be
expressed when additional deletions are made in the adenovirus
genome. For example, adenoviruses deleted in both E1 and E3 regions
are capable of carrying up to 10 kB of foreign DNA and can be grown
to high titers in 293 cells (Stratford-Perricaudet and Perricaudet,
1991. An embodiment includes substitution of a thiol-containing
compound transporter gene or segment of a thiol-containing compound
transporter gene under the control of the minimum amount of
replication-incompetent adenovirus.
[0278] Other viral vectors may be employed as expression
constructs. Vectors derived from viruses such as vaccinia virus
(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)
adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden,
1986; Hermonat and Muzycska, 1984) and herpes viruses may be
employed. They offer several attractive features for various
mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and
Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
[0279] Several non-viral methods for the transfer of expression
vectors into cultured mammalian cells also are contemplated. These
include calcium phosphate precipitation (Graham and Van Der Eb,
1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran
(Gopal, 1985), lipofectamine-DNA complexes, and receptor-mediated
transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these
techniques may be successfully adapted for in vivo or ex vivo
use.
[0280] Insertional variants include fusion proteins such as those
used to allow rapid purification of the polypeptide and also may
include hybrid proteins containing sequences from other proteins
and polypeptides that are homologues of the polypeptide. For
example, an insertional variant may include portions of the amino
acid sequence of the polypeptide from one species, together with
portions of the homologous polypeptide from another species. Other
insertional variants may include those in which additional amino
acids are introduced within the coding sequence of the polypeptide.
These typically are smaller insertions than the fusion proteins
described above and are introduced, for example, to disrupt a
protease cleavage site.
[0281] Another method for the preparation of the polypeptides use
peptide mimetics. Mimetics are peptide-containing molecules that
mimic elements of protein secondary structure. (Johnson et al.,
"Peptide Turn Mimetics" in Biotechnology and Pharmacy, Pezzuto et
al., Eds. Chapman and Hall, New York (1993)). The underlying
rationale behind the use of peptide mimetics is that the peptide
backbone of proteins exists chiefly to orient amino acid side
chains in such a way as to facilitate molecular interactions, such
as those of antibody and antigen. A peptide mimetic is expected to
permit molecular interactions similar to the natural molecule (i.e.
transporting thiol-containing compounds to the outside of the
cell). An embodiment includes the use of protein mimetics to mimic
the excretion of thiol-containing compounds within a given
transporter responsible for thiol-containing compound
transport.
[0282] Successful applications of the peptide mimetic concept have
thus far focused on mimetics of .beta.-turns within proteins, which
are known to be highly antigenic. Once the component amino acids of
the turn are determined, peptide mimetics may be constructed to
achieve a similar spatial orientation of the essential elements of
the amino acid side chains.
[0283] The embodiments are generally directed to compositions and
methods for the treatment of diseases characterized by defective
thiol-containing compound transport in tissues (i.e. including
cystic fibrosis, and diseases with excessive accumulation of mucus,
including cystic fibrosis, chronic bronchitis and asthma). It has
been found, within the context, that certain agents (i.e. flavones,
isoflavones, flavanones, isoflavanones) are capable of stimulating
thiol-containing compound transport in tissues (i.e. epithelial
tissues of the airways, intestine, pancreas and other exocrine
glands). Such therapeutic compounds may be administered to patients
afflicted with a thiol-containing compound transporter deficiency
as described herein.
Compound Analysis
[0284] In a preferred embodiment, a nucleic acid may include a
recombinant expression vector containing nucleic acid with a
nucleotide sequence including a thiol-containing compound
transporter. Preferably, a cell into which the nucleic acid is
transfected is deficient in thiol-containing compound transport so
that the effects of a potential activator are assessed in the
presence of a single, isolated thiol-containing compound
transporter confering protein. In another preferred embodiment a
therapeutic agent and substance to be tested are incubated in
culture with the cell and the level of thiol-containing compounds
measured in the extracellular media. Alternatively, the cell can be
a thiol-containing compound transporter cell in a transgenic
animal, transgenic for a nucleic acid, and the therapeutic agent
and substance to be tested are administered to the transgenic
animal. Furthermore, the cell can be a cell in culture isolated
from a thiol-containing compound transporter transgenic animal. The
sensitivity of the cell for the therapeutic agent in the presence
and absence of the potential therapeutic agent is assessed by
determining the concentration of the therapeutic agent that exports
a predetermined level of the thiol-containing compound from the
cell either in the presence or in the absence of the substance
being tested. Once an agent provides positive results on the
cellular level and the thiol-containing compound is verified by a
measuring device for example an HPLC, a cell system utilizing a
membrane to separate the basolateral and apical sides of a cellular
monolayer may be used to further test transporter stimulation to
release thiol-containing compounds. In addition, if the agent
demonstrates positive affects on apical transport of
thiol-containing compounds, these agents may be further tested in
an animal model for example the mouse lung as described.
[0285] One embodiment includes a method for identifying a substance
that directly increases the synthesis and/or activity of a
thiol-containing compound transporter involving incubating a
substance to be tested with a cell and determining the amount of
thiol-containing compound in the media.
[0286] In one embodiment, anti-thiol-containing compound
transporter antibodies labelled with a detectable substance, such
as a fluorescent marker, an enzyme or a radioactive-marker may be
used to identify cells expressing a thiol-containing compound
transporter. Tissue removed from a patient may be used as the cell
sample. A tissue section, for example, a freeze-dried or fresh
frozen section of tissue (i.e. lung tissue) removed from a patient,
may also be used as the sample. The samples can be fixed and the
appropriate method of fixation may be chosen depending upon the
type of labelling used for the antibodies. Alternatively, a cell
membrane fraction can be separated from the tissue removed from a
patient and can be used as the sample. Conventional methods such as
differential or density gradient centrifugation can be used to
separate out a membrane fraction.
[0287] A thiol-containing compound transporter cell may be
identified by incubating an antibody, for example a monoclonal
antibody, with a cell to be tested for thiol-containing compound
transporter. Binding of the antibody to the cell is indicative of
the presence on the cell of a protein having thiol-containing
compound transporter activity. The level of antibody binding to the
cell can be compared to the level of antibody binding to a normal
control cell, and increased binding of the antibody to the cell as
compared to the normal cell can be used as an indicator of increase
expression of a thiol-containing compound transporter. Binding of
an antibody to a cell (i.e. a cell to be tested or a normal control
cell such as a cell from a condition-free patient) may be
determined by detecting a substance with which the antibody is
labelled. The detectable substance may be directly coupled to the
antibody, or alternatively, the detectable substance may be coupled
to another molecule that can bind the antibody (i.e. a secondary
antibody or anti-antibody).
[0288] A thiol-containing compound transporter cell can be detected
as described above in vitro in a sample prepared as described
above. For example, a section on a microscope slide can be reacted
with antibodies using standard immunohistochemistry techniques
[0289] Additionally, if a single cell suspension of cells can be
achieved, the cells can be reacted with antibody and analyzed by
flow cytometry. Alternatively, a thiol-containing compound
transporter cell can be detected in vivo in a subject bearing a
thiol-containing compound transporter deficiency. Labelled
antibodies can be introduced into the subject and antibodies bound
to the tissue can be detected. For example, the antibody can be
labelled with a radioactive marker whose presence and location in a
subject can be detected by standard imaging techniques.
[0290] The antibodies, and compositions thereof, may also be used
to inhibit the non-thiol-containing compound transporter component
of a cell. The embodiments provide a method for inhibiting the
non-thiol-containing compound transporter region of protein in a
cell comprising inhibiting activity of a protein. Preferably, the
thiol-containing compound transporter cell is a lung cell. A
thiol-containing compound transporter can increase its
thiol-containing compound transport by interfering with the
non-thiol-containing compound transporter activity of the protein.
For example, the ability of a thiol-containing compound transporter
protein to transport non-thiol-containing compounds may be
impaired. Accordingly, any molecule which binds to a protein having
thiol-containing compound transporter activity and whose binding
inhibits the non-thiol-containing compound transporter activity of
the protein are encompassed by invention.
[0291] The methods for increasing the activity of thiol-containing
compound transporters and/or the synthesis of thiol-containing
compound transporter proteins and/or thiol-containing compound
transporter and/or transfection of a thiol-containing compound
transporter gene can be applied to patients having a
thiol-containing compound transporter deficiency. The compositions
and methods can be particularly useful in treating for example lung
(i.e. CF), pancreatic, gastrointestinal, vascular, joint,
neurodegenerative and biliary diseases and also male
infertility.
[0292] One embodiment also provides a diagnostic kit for
identifying an agent that increases thiol-containing compound
transport protein activity and/or expression comprising an agent, a
cell and a means for detecting thiol-containing compounds,
thiol-containing compound transporter protein; means for
determining the amount of protein in the sample; and means for
comparing the amount of protein in the sample with a standard.
Preferably, the molecule is a monoclonal antibody. Other molecules
that can bind a protein having thiol-containing compound
transporter activity can be used, including the bispecific
antibodies and tetrameric antibody complexes. The diagnostic kit
can also contain an instruction manual for use of the kit.
[0293] Effects of replenishing thiol-containing compounds in site
specific compartments. In one embodiment, cells are treated with
one or more agents to increase the transport of thiol-containing
compounds (for example, glutathione) into the mitochondria. Loss of
CFTR function is associated with diminished mitochondria
glutathione levels (see FIG. 11). This may be due to the effects of
CFTR directly or indirectly on the mitochondrial glutathione
transporter(s). The loss or dysfunction of these mitochondrial
glutathione transporters produces a mitochondrial oxidative stress
(see FIG. 9). Again, a CFTR defect leads to similar decreases in
mitochondrial glutathione levels as seen in the ELF and suggests
that other ABC transporters may also be involved and can be used to
replenish glutathione transport. A number of diseases have been
associated with mitochondrial oxidative stress and include
alcoholism and associated disease such as hepatitis and cirrhosis,
neurodegenerative diseases (such as Parkinsonism, Alzhiemers, and
Hunnigton's Disease), inheritable disorders such as myopathy,
chronic alcoholism, optic atrophy, dystonia, Leigh's syndrome,
myoclonic epilepsy and ragged red fiber (MERRF), mitochondrial
encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS)
and diabetes. Any one of these conditions may be a target for
treatment by one or more of the disclosed agents to increase the
transport of thiol-containing compounds to the mitochondria.
[0294] In the foregoing specification, the embodiments have been
described with reference to specific exemplary embodiments. It
will, however, be evident that various modifications and changes
may be made without departing from the broader spirit and scope as
detailed in the appended claims. The specification and figures are,
accordingly, to be regarded in an illustrative rather than a
restrictive sense.
[0295] In several embodiments, the restoration of GSH levels has
been described. In particular embodiments restoration of lung GSH
levels in compromised patients has been described. Some of these
embodiments include the restoration of GSH in the mitochondria of
the lung thus likely relieving mitochondrial oxidative stress and
may also alleviate the exacerbated response to infection-induced
inflammation.
[0296] The embodiments are further illustrated by the following
examples and detailed protocols. However, the examples are merely
intended to illustrate embodiments and are not to be construed to
limit the scope herein. The contents of all references and
published patents and patent applications cited throughout this
application are hereby incorporated by reference.
EXAMPLES
Example 1
FIG. 5--Reduced Glutathione (GSH) Concentrations in Mouse
Epithelial Lining Fluid (ELF)
[0297] GSH concentrations in ELF were calculated from GSH
concentrations in bronchoalveolar lavage fluid (BALF). Briefly, a
lung was lavaged through a tracheal canula with three separate 1 mL
aliquots of phosphate-buffered saline (pH 7.4). Each aliquot was
instilled into the lung and withdrawn only once. All three aliquots
were then pooled and centrifuged at 4000.times.g to remove cells
(i.e., alveolar macrophages). The cell-free BALF was acidified with
metaphosphoric acid to a final concentration of 0.75%
metaphosphoric acid and centrifuged at 10,000.times.g to pellet the
precipitated proteins. GSH concentrations were determined
spectrophotometrically with a commercially available assay that
forms a chromogen with GSH. ELF concentrations of GSH were
calculated from the BALF concentrations multiplied by a dilution
factor derived from the difference in serum and BALF urea
concentrations. GSH concentrations in ELF of cystic fibrosis
transmembrane regulator protein knockout (CFTR-KO) mice were lower
(249.+-.59 .mu.M) compared to wild type mice (512.6.+-.63 .mu.M).
Data are shown as the mean.+-.standard error for n.gtoreq.5 and
significance (*) attained at p.ltoreq.0.05.
Example 2
[0298] FIG. 6 Glutathione Reductase (GR) Activity in Lung Tissue of
Wild Type and Cystic Fibrosis Transmembrane Regulator Protein
Knockout (CFTR-KO) Mice. Mouse lung tissue (10-25 mg) were ground
in liquid nitrogen and dissolved in 800 .mu.L of cold
homogenization buffer (50 mM potassium phosphate, 1 mM EDTA, pH
7.5). The sample was centrifuged at 8,500.times.g for 10 minutes at
4.degree. C. and the supernatant retained for analysis. GR activity
in the supernatant was determined spectrophotometrically (340 nm)
from the rate of NADPH consumption by GR in the presence of
oxidized glutathione (GSSG) using a commercially available kit. GR
is expressed as units per milligram of protein in the supernatant.
GR activity in CFTR-KO mouse lungs was significantly elevated
(3.76.+-.0.27 U/mg protein) compared to WT mouse lungs
(2.54.+-.0.19 U/mg protein). Data are shown as the mean.+-.standard
error for n.gtoreq.12 and significance (*) attained at
p.ltoreq.0.05.
Example 3
FIG. 7--Glutathione Peroxidase (GPx) Activity in Lung Tissue of
Wild Type and Cystic Fibrosis Transmembrane Regulator Protein
Knockout (CFTR-KO) Mice
[0299] Mouse lung tissue (10-35 mg) was ground in liquid nitrogen
and the ground tissue dissolved in 1.0 mL of cold homogenization
buffer (50 mM Tris-HCl, 5 mM EDTA and 1 mM 2-mercaptoethanol, pH
7.5). Homogenate was centrifuged (7,500.times.g, 15 min., 4.degree.
C.) and the supernatant retained for analysis. The GPx activity in
the sample was determined from a commercially available kit to
which t-butyl-hydroperoxide was added as a GPx substrate to
generate oxidized glutathione (GSSG). The rate of NADPH consumption
by glutathione reductase in the subsequent reduction of GSSG was
used to calculate GPx activity. GPx activity was normalized to
sample protein concentrations. CFTR-KO mice had significantly more
GPx activity (431.+-.28 U/mg protein) in the lung tissue than WT
mice (338.+-.20 U/mg protein). Data are shown as the
mean.+-.standard error for n.gtoreq.10 and significance (*)
attained at p.ltoreq.0.05.
Example 4
FIG. 8--Concentration of 8-hydroxy-2'-deoxyguanosine (8OH2dG) in
Lung Tissue of Wild Type (WT) and Cystic Fibrosis Transmembrane
Regulator Protein Knockout (CFTR-KO) Mice
[0300] DNA from WT and CFTR-KO was obtained by a chloroform-isoamyl
alcohol extraction of proteinase K-digested lung homogenates. The
purified DNA was subsequently hydrolyzed to nucleosides with
nuclease P1 and alkaline phosphatase. Samples were analyzed for
8OH2dG and 2-deoxyguanosine (2dG) by HPLC coupled with
electrochemical and UV detectors. To normalize for differences in
DNA yield between lung samples, the ratio of 8OH2dG to 10.sup.5 2dG
were calculated. Levels of 8OH2dG/10.sup.5 2dG were significantly
increased in CFTR-KO lungs (5.67.+-.0.94) compared to WT mice
(172.+-.0.37). Data are shown as the mean.+-.standard error for n 8
and significance (*) attained at p.ltoreq.0.05.
Example 5
FIG. 9 Mitochondrial Aconitase activity in the Lungs of Wild Type
and Cystic Fibrosis Transmembrane Regulator Protein Knockout
(CFTR-KO) Mice
[0301] Aconitase is a mitochondrial and cytosolic enzyme that is
sensitive to oxidative stress. A loss of aconitase activity in
isolated mitochondria can be used as a direct indicator of
mitochondrial oxidative stress. Mitochondria from the lungs of wild
type (control) and CFTR-KO mice were obtained by differential
centrifugation of lung homogenates. Briefly, lungs were homogenized
in mitochondrial isolation buffer (210 mM mannitol, 70 mM sucrose,
5 mM Tris-HCl, 1 mM EDTA, pH 7.5) and cellular debris removed by
repeated centrifugations at 1,300.times.g until no pellet was
obtained. The supernatant was then centrifuged at 17,000.times.g to
pellet mitochondria. The mitochondria were then resuspended in a
small volume of mitochondria lysis buffer (cysteine 1 mM, citric
acid 1 mM, Triton X-100 0.5%, pH 7.4) and assayed for aconitase
activity. Aconitase activity was determined spectrophotometrically
by following the formation of cis-aconitate from isocitrate at 240
nm. Mitochondrial aconitase activity was significantly decreased in
CFTR-KO lungs (63.1.+-.10.2 U/mg protein) compared to WT lungs
(119.6.+-.8.8 U/mg protein). Data are shown as the mean.+-.standard
error for n and significance (*) attained at p.ltoreq.0.05.
Example 6
FIG. 10 Concentration of Lipid Peroxidation in Lungs of Wild Type
(WT) and Cystic Fibrosis Transmembrane Regulator Protein Knockout
(CFTR-KO) Mice
[0302] Approximately 25 mg of lung tissue were homogenized in 50 mM
phosphate buffer containing 1 mM butylated hydroxytoluenc and
acidified with an equal volume of phosphoric acid. Thiobarbituric
acid is known to reactive with oxidized lipid breakdown products
and is a commonly used marker for lipid peroxidation.
Thiobarbituric acid was added and the mixture heated at 90.degree.
C. for 45 minutes. The chromogen was extracted with n-butanol and
the absorbance at 535 nm measured. TBARS concentrations were
calculated from a standard curve, normalized for sample protein and
presented as the % change from control (WT) arbitrarily set at
100%. Levels of TBARS in CFTR-KO mouse lungs were significantly
increased (126.6.+-.8.0%) compared to WT controls (99.85.+-.4.1%).
Data are shown as the mean.+-.standard error for n 6 and
significance (*) attained at p 0.05.
Example 7
FIG. 11 Mitochondrial Glutathione (GSH) Concentrations in the Lung
and Small Intestine of Wild Type and Cystic Fibrosis Transmembrane
Regulator Protein Knockout (CFTR-KO) Mice
[0303] Mitochondria were isolated from homogenized lung and small
intestine by differential centrifugation. Briefly, lungs and small
intestines were homogenized in mitochondrial isolation buffer (210
mM mannitol, 70 mM sucrose, 5 mM Tris-HCl, 1 mM EDTA, pH 7.5) and
cellular debris removed by repeated centrifugations at
1,300.times.g until no pellet was obtained. The supernatant was
then centrifuged at 17,000.times.g to pellet mitochondria. Isolated
mitochondria were then resuspended in phosphate-buffered saline and
protein concentrations determine. Mitochondria were lysed by the
addition of metaphosphoric acid (final concentration of 1%) and
precipitated proteins pelleted by centrifugation at 20,000.times.g.
GSH concentrations were determined by HPLC coupled with
electrochemical detection and normalized to the protein
concentration. The wild type mice (C57BL/6) have functional CFTR in
both the lungs and intestinal tract. The C57BL/6 CFTR-KO mice do
not have functional CFTR in either the lungs or the intestinal
tract. In the FABP-Tg CFTR-KO mice, a functional CFTR protein has
been restored to the intestinal tract. Mitochondrial GSH
concentrations in the lungs of both the CFTR-KO lines were
significantly lower than wild type mice. In the small intestine of
the C57BL/6 CFTR-KO mice, mitochondrial GSH concentrations were
significantly lower than the wild type mice. In the FABP-Tg CFTR-KO
mice where intestinal expression of CFTR has been restored,
mitochondrial GSH concentrations were not significantly different
than wild type mice. Lung mitochondrial GSH concentrations in the
FABP-Tg CFTR-KO mice, however, still remained significantly lower.
Data are shown as the mean.+-.standard error for n 6 and
significance from wild type (*) lung and (**) intestine attained at
p 0.05.
Example 8
FIG. 12 Defective Cystic Fibrosis Transmembrane Regulator (CFTR)
Protein and Potential Pathways to Lung Disease
[0304] Defective CFTR activity in the lung results in decreased GSH
transport in the lung. Across the epithelial surface, decreased GSH
transport via CFTR will produce a concomitant decrease in the
concentration of GSH in the epithelial lining fluid (ELF) that
covers the airspace surface. In addition, defective CFTR produces a
decrease in mitochondrial GSH concentrations. Whether this
consequence is due to a direct CFTR effect on mitochondrial GSH
transport or through a secondary pathway is unclear. The decrease
in ELF GSH concentrations may impair lung defense mechanisms and
permit persistent and recurring lung infections. Mitochondrial
oxidative stress, primarily through superoxide leaking from
oxidative phosphorylation, is increased because GSH concentrations
are decreased. Taken together, defective GSH transport from the
CFTR mutation may initiate the oxidative stress from the chronic
infections and mitochondria that produces a progressive
deterioration of the lung structure and function resulting in
pulmonary failure and death.
Example 9
FIG. 14--Cellular Synthesis, Metabolism and Transport of
Glutathione (GSH)
[0305] GSH is synthesized from its constituent amino acids (L-Glu,
L-Cys, and L-Gly) by the sequential action of
.gamma.-glutamylcysteine synthetase (.gamma.GCS) and GSH synthetase
(GS). Steady state GSH levels reflect a balance between synthesis,
consumption and transport. GSH can reduce deleterious peroxides by
action of glutathione peroxidase (GPx) to generate oxidized
glutathione (GSSG). Once oxidized, GSSG can be reduced back to GSH
by glutathione reductasc that derives its reducing equivalents from
NADPH. Certain members of the ABC transporter family can transport
GSH across the cell membrane. In the apical membrane of a pulmonary
epithelial cell, these transporters may include the cystic fibrosis
transmembrane regulator protein (CFTR) and multidrug resistance
proteins 2 and 4 (MRP2 and MRP4 respectively). CFTR, and
potentially the MRP proteins, play an important role in the
maintenance of epithelial lining fluid (ELF) GSH concentrations.
ELF GSH can be recycled by the coordinated activity of
.gamma.-glutamyltransferase (GGT) and dipeptidase (DP) that cleave
GSH into its amino acid constituents and transfer them into the
cytoplasm.
Example 10
FIG. 15--Pseudomonas Killing by an Eight-Hour Exposure to Mouse
Bronchoalveolar Lavage Fluid (BALF)
[0306] Pseudomonas aeruginosa was cultured in the presence of
increasing concentrations of BALF for 8 hours and pseudomonas
viability then determined by Colony Forming Units (CFU) on agar
plates. BALF was obtained through a tracheal canula. Two separate 1
mL aliquots of phosphate-buffered saline (PBS; pH 7.4) were
instilled into the lung and withdrawn. The aliquots were then
pooled and centrifuged at 4000.times.g to remove cells (i.e.,
alveolar macrophages). Bacteria were then exposed to PBS control
(ctr), 5%, or 25% dilution of HALF for 8 hours. Following the
exposure, bacteria were then cultured and the number of CFU
determined. The greater the antibacterial properties of the
exposure condition the less CFUs. Exposure of pseudomonas to 25%
BALF greatly decreased 50%) the number of pseudomonas CFU. This
demonstrates that BALF contains antibacterial modulators.
Example 11
[0307] FIG. 16 Extracellular Concentration of Glutathione (GSH)
from Rutin and Dexamethasone Treated Cells.
[0308] Cells were treated with various concentrations of rutin or
dexamethasone for 48 hours and then the GSH concentration in the
media determined. Cells, CFTR-deficient CRL-1687 cells, were grown
to approximately 90% confluency in 24-well plates and then exposed
to media containing the varying concentrations of rutin or
dexamethasone. At 48 hours the media was removed and GSH
concentrations determined by HPLC coupled with electrochemical
detection and normalized to the protein concentration.
Example 12
FIG. 17 Dexamethasone-Induced Changes in Epithelial Lining Fluid
(ELF) Glutathione (GSH) Concentrations
[0309] Wild type (C57/B6) mice were given a 1 mg/kg dexamethasone
(DEX) injection (intraperitoneal) daily for two days.
Bronchoalveolar lavage fluid (BALF) was obtained at 48 hours after
the first initial dose and GSH concentrations determined. BALF was
obtained through a tracheal canula with a single 2.0 mL aliquots of
phosphate-buffered saline (pH 7.4) that was instilled into the lung
and withdrawn only once. The BALF was centrifuged at 4000.times.g
to remove cells (i.e., alveolar macrophages). The cell-free BALF
was acidified with metaphosphoric acid to a final concentration of
1% metaphosphoric acid and centrifuged at 20,000.times.g to pellet
the precipitated proteins. GSH concentrations were determined by
HPLC coupled with electrochemical detection. ELF concentrations of
GSH were calculated from BALF concentrations multiplied by a
dilution factor derived from the difference in serum and BALF urea
concentrations. ELF concentrations in DEX treated mice were
significantly increased (75.3.+-.12.0 .mu.M) compared to untreated
mice (18.9.+-.2.3 .mu.M). Data are shown as the mean.+-.standard
error for n.gtoreq.4 with significance attained at p S 0.05.
Example 13
FIG. 18 Epithelial Lining Fluid (ELF) Glutathione (GSH)
Concentrations and Lung MRP2 and CFTR Expression in Pseudomonas
infected Wild Type Mice
[0310] Wild type (C57/B6) mice were infected with Pseudomonas
aeruginosa via intratracheal instillation of Pseudomonas-coated
particles. Forty-eight hours following the inoculation,
bronchoalveolar lavage fluid (BALF) and lung tissue were harvested.
ELF concentrations of GSH were calculated from BALF concentrations
multiplied by a dilution factor derived from the difference in
serum and BALF urea concentrations. Lungs were lavaged through a
tracheal canula with three separate 1 mL aliquots of
phosphate-buffered saline (pH 7.4). Each aliquot was instilled into
the lung and withdrawn only once. All three aliquots were then
pooled and centrifuged at 4000.times.g to remove cells (i.e.,
alveolar macrophages). The cell-free BALF was acidified with
metaphosphoric acid to a final concentration of 0.75%
metaphosphoric acid and centrifuged at 20,000.times.g to pellet the
precipitated proteins. GSH concentrations were determined
spectrophotometrically with a commercially available assay that
forms a chromogen with GSH. Lungs from Pseudomonas infected mice
were homogenized in membrane isolation buffer (250 mM sucrose, 10
mM Tris-HCl, pH 7.5; MIB) and filtered through silk to remove large
debris. Homogenate was then centrifuged at 33,000.times.g to pellet
membranes. Membranes were resuspended in MIB for Western blotting.
Membrane proteins (30 .mu.g) were separated on 8% agarose gels,
transferred to PVDF membranes for determination of MRP2 and CFTR
expression. GSH concentrations in the ELF of pseudomonas infected
mice (1282.+-.238 .mu.M) were significantly elevated compared to
uninfected control mice (201.+-.75 .mu.M). Data are shown as the
mean.+-.standard error for n=5 with significance attained at p
0.05. Western blots demonstrate an increase in the expression of
both MRP2 and CFTR in pseudomonas infected lungs compared to
uninfected mice.
Techniques used in Experimentation
[0311] Serum and BALF Urea Concentrations.
[0312] To determine actual ELF concentrations of soluble
antioxidants from BALF, a dilution factor is derived from the
difference between BALF and serum urea concentrations. The
assumption that urea freely diffuses between the vascular and ELF
compartments are used as an indicator of ELF dilution. (Rennard, S.
I. Estimation of volume of epithelial lining fluid recovered by
lavage using urea as a marker of dilution, J. of Applied Physiol.
1986 vol. 60 532-550). A dilution factor is thereby obtained by
dividing the serum urea concentration by the BALF concentration.
ELF concentrations are then calculated by multiplying the BALF
concentrations by the dilution factor. Urea concentrations in the
samples are determined using a commercially available reagent
(Sigma Diagnostics 66-20; St. Louis, Mo.).
[0313] Western Blot.
[0314] Lung apoprotein levels of CFTR, MRP-2 and MDR-1 may be
determined by western blot analysis. Frozen and fresh lung tissue
will be homogenized and 10-30 .mu.g of protein separated by
SDS-PAGE (8% acrylamide gels) on a mini protean-3 electrophoresis
system at 100 V. Proteins will be transferred onto PVDF membrane
and blocked overnight at 4.degree. C. with 5% horse serum in Tris
balanced salt solution with tween-20 (TBS-T). Proteins will be
identified using commercial antibodies against CFTR (monoclonal
24-1, R&D Systems), MRP-2 (monoclonal M.sub.2111-6, Alexis) and
MDR-1 (monoclonal 265/F4, NeoMarkers) as primary antibodies that
are incubated at room temperature for 2-3 hours, washed extensively
with TBS-T and then incubated with the appropriate secondary rabbit
antimouse or other antibody conjugated with HRP for 30 minutes at
room temperature. Blots are then extensively washed with TBS-T and
developed with an ECL chemiluminescence kit (Amersham) and captured
on X-ray film. Densitometry is performed with a gel imaging system
(CDD Bio, Hitachi).
[0315] RT-PCR Analysis.
[0316] RT-PCR will be performed using Advantage one-step kit with a
RT-PCR control amplimer set containing mouse G3PDH (Clontech). The
primer sequences for the mouse MDR1 gene
(5'-CTCACCAAGCGACTCCGATACATG-3'; 5'-GATAATTCCTGTGCCAAGGTTTGCTAC-3')
and (5'-AAGACAAAGATTCTAGTGTTGGACG-3');
(5'-AGATATGCCAGAGATCAGTTCACACC-3') for the MRP-2 gene will be used
as described(50). RT-PCR products are visualized by UV illumination
after electrophoresis through 2% agarose gels and documented using
the gel imaging system (CCD Bio, Hitachi).
[0317] Immunocytochemistry.
[0318] An immunoperoxidase method (Oury et.al 1994.
"Immunocytochemical localization of extracellular superoxide
dismutase in human lung". Lab Invest. 70:889-898) will be used for
light microscopic immunocytochemical labeling. Tissue nonspecific
binding to antibodies is blocked by incubation with 5% normal goat
serum, 5% gelatin and 1% BSA. Sections are then incubated with
either the pre-immune serum or the primary antibody against either
MRP-2 or MDR-1 in 0.1% gelatin and 1% BSA in PBS for 1 hour at room
temperature. They are washed and incubated with biotin labeled
rabbit anti-mouse diluted in 0.1% gelatin and 1% BSA for 1 hour.
The labeling signals are intensified by incubation with
streptavidin conjugated to horseradish peroxidase in 0.1%
gelatin+1% BSA. Labeling is detected by incubating in
diaminobenzidine (10 mg diaminobenzine, 50 ml 0.05 M Tris Cl, pH
7.6, 100 .mu.l 3% H.sub.2O.sub.2). After the incubation, slides are
counter stained with 1% methyl green, washed, dehydrated in
ethanol, cleared with xylene and mounted in flowtek.
[0319] 5. Aconitase Activity.
[0320] Aconitase inactivation is a sensitive marker for superoxide
or peroxynitrite formation in the mitochondria. Aconitase activity
is measured spectrophotometrically by monitoring the formation of
cis-aconitate from isocitrate at 240 nm as previously described by
Patel et.al. 1996 "Requirement for superoxide in excitotoxic cell
death. Neuron 16:345-355)
[0321] 6. F2-Isoprostane Formation.
[0322] The formation of F2-isoprostanes will be measured by GC/MS
(gas chromatography/mass spectroscopy) as previously described.
Briefly, F2-isoprostanes will be extracted from tissue with
chloroform/methanol (2:1, v/v) containing 0.005% butylated
hydroxytoluene and the organic phase evaporated to dryness under
vacuum. The F2-isoprostanes will be released from the lipids by
hydrolysis in 4 ml methanol plus 4 ml KOH (15%) at 37.degree. C.
for 30 minutes. The free isoprostanes are derivatized with
N,O-bis(trimethylsilyl)trifluoroacetamide and F2-isoprostanes
quantified by gas chromatography/negative ion chemical ionization
mass spectrometry (GC/NICl-MS) using [.sup.2H.sub.4]-PGF2a (Cayman
Chemical, Ann Arbor, Mich.) as an internal standard.
[0323] 7. HPLC Analysis for 8-Hydroxy-2-Deoxyguanosine in Lung
DNA:
[0324] DNA from mouse lung tissue was obtained by a
chlorofomilisoamyl alcohol extraction of proteinase K digested lung
homogenates. The purified DNA was then hydrolyzed to nucleosides
with nuclease P1 and alkaline phosphatase. Samples were analyzed
for 8-hydroxy-2-deoxyguanosine (8OH2dG) and 2-deoxyguanosine (2dG)
by HPLC coupled with coulometric electrochemical and UV detection
(CoulArray Model 5600; ESA Inc., Chelmford, Mass.) for 8OH2dG and
2dG respectively. Sample analysis was done using a 4.6.times.150
mm, C-18 reverse phase column (YMCbasic.RTM.; YMC Inc., Wilmington,
N.C.) with a mobile phase of 100 mM sodium acetate in 5% methanol
at pH 5.2. UV detects 2dG at 265 nm while 8OH2dG was detected
electrochemically with electrode potentials of 285, 365 and 435 mV.
Under these conditions, 2dG and 8OH2dG had retention times of
approximately 7.4 and 9.5 minutes respectively. Nucleoside
concentrations were calculated from standard curves generated daily
with freshly prepared standards.
[0325] 8. Pseudomonas Killing Assay.
[0326] This assay was derived from an assay used to study bacterial
killing by neutrophils (Hampton, M. B. 1999 "Methods for
quantifying phagocytosis and bacterial killing by human
neutrophils" J. Immunol. Methods 232:15-22). Inoculums of
Pseudomonas aeruginosa (PA01) are grown in LB media in the presence
or absence of mouse BALF and viability assessed at various time
points (usually 4 to 8 hours). Mice are anesthetized with
pentobarbital followed by exsanguinations by direct cardiac
puncture.
[0327] Approximately 1 ml of blood is collected in heparinized
tubes and plasma prepared by centrifugation and stored at
-80.degree. C. until use. BALF was collected using one 1-mL aliquot
of sterile phosphate buffered, pH 7.4 (PB). The aliquot is
centrifuged (2000.times.g for 5 minutes at 4.degree. C.) to recover
cells. An aliquot of the cell free BALF supernatant is acidified
with 5% In-phosphoric acid and the supernatant retained and stored
at -70.degree. C. for subsequent analyses. The remaining cell free
BALF is used for testing in its effect in host defense in the
Pseudomonas growth inhibition assay. The right and left lungs are
then removed and either quick-frozen in liquid nitrogen or fixed
for immunocytochemistry.
[0328] To minimize GSH loss during the evaluation, BALF is
acidified with 5% m-phosphoric acid (150 .mu.L/mL), cooled on ice
and centrifuged (10,000.times.g for 10 min. at 4.degree. C.) to
remove precipitated proteins. The lung tissue, approximately 20 mg
of the ground tissue is dissolved in 600 .mu.L of PBS, acidified
with 50 .mu.L of 5% m-phosphoric acid, cooled on ice, and
centrifuged (10,000.times.g for 10 min. at 4.degree. C.) to remove
precipitated proteins. GSH and GSSG in BALF and tissues are
analyzed by HPLC coupled with coulometric electrochemical detection
(CoulArray Model 5600; ESA Inc., Chelmford, Mass.). Sample analysis
was done using a 7.times.53 mm C-18 reverse phase (Platinum EPS C18
100 A 3 .mu.m, Alltech Associates Inc., Deerfield, Ill.) and a
mobile phase of 125 mM potassium acetate in 1% acetonitrile at pH
of 3.0. The electrode potentials in a four-channel electrode array
were set at 100, 215, 485 and 570 mV. Under these conditions, GSH
exhibited a retention time of 2.7 minutes with a signal distributed
across channels 2, 3 and 4; GSSG exhibited a retention time of 4.2
minutes with a signal confined to channel 4. Concentrations of GSH
and GSSG from a 104 injection can be determined from a five-point
calibration curve generated from standards prepared fresh
daily.
[0329] Bacterial viability is assessed by the loss of ability of
bacteria to form colonies after plating on nutrient agar. In
preliminary studies it was determined that a 1:2,000,000 dilution
of the culture plated on agar overnight gives a good number of CFUs
(colony forming units) with the PA01 strain (see preliminary data).
To control for dilutional effects of the BALF we control with the
same % of PBS. This assay provides us with a functional endpoint to
assess changes in host defense of the BALF.
[0330] 9. Cytokine Analyses.
[0331] Levels of TNF-.alpha., MiP-2 and IL-10 will be measured on
BALF using commercial ELISA kits (MTAOO, MM200 & M1000, R&D
Systems, Minneapolis, Minn.). These cytokines are surrogate markers
of inflammation. TNF-.alpha. and MIP-2 serve as pro-inflammatory
cytokine markers and IL-10 serves as an anti-inflammatory cytokine
marker. These markers are either elevated (TNF-.alpha., MIP-2) or
depressed (IL-10) in CF BALF.
[0332] 10. Histopathology.
[0333] Mice from each group will be used for histopathology. Mice
will be anesthetized with avertin and their trachea cannulated and
instilled with 2% paraformaldehyde plus 2% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.4. After 10 minutes of fixation within the
chest cavity, lungs are removed and 2 mm slices are cut and
immersion fixed in 4% paraformaldehyde for overnight fixation and
embedded in parafilm. For electron microscopy, 2 mm slices are
placed in 2% glutaraldehyde for four hours then cubed into
2.times.2.times.2 blocks and washed in cacodylate buffer and
post-fixed with 2% OsO.sub.4. The blocks are dehydrated in a graded
series of ethanol, transferred to propylene oxide, and embedded in
Epox. A diamond knife is used to cut thin sections and placed on a
200 mesh uncoated grid. Sections are stained with uranyl acetate
and lead citrate prior to viewing. Sections will be viewed for both
extent and severity of tissue injury and inflammatory cell
infiltration.
TABLE-US-00002 TABLE 2 Proposed Apical Lung Glutathione
Transporters. Trans- Tissue Inducers & porter expression
Activators Inhibitors MDR-1 Lung, Kidney, Dexamethasone(I),
Apigenin, Liver, GI Genistein(A), Reserpine, Quercetin(A) PSC 833,
Diltazem, Verapamil, Acridine Orange MRP-2 Lung, Liver,
Dexamethasone(I), Genistein, GI Quercetin(A), Phenobarbital,
Cisplatin(A), Probenecid, Indomethacin(A) benzbromarone
Glibenclamine, MK-571, Indocyanine Green MRP-4 Lung, GI, Unknown
Unknown Pancreas, Muscle CFTR Lung, GI, S-nitrosoglutathione(I),
Genistein, Pancreas Ibuprofen(A), glibenclamine Genistein(A),
Apigenin(A), Quercetin(A) (I)Inducer (A)Activator
TABLE-US-00003 TABLE 3 Concentration % Increase Over Drug (.mu.M)
Control * p-Aminosalicylic Acid 100 414 .sup.a Berberine 100 170
.sup.a Biochanin-A 50 133 .sup.c Dexamethasone 50 178 .sup.a
Diltiazem 100 119 .sup.b Indomethacin 100 235 .sup.a
Methylsalicylic Acid 100 169 .sup.a Propyl Gallate 50 136 .sup.a
Rutin 100 160 .sup.b Sulfasalazine 50 211 .sup.a 5-Sulfosalicylic
Acid 50 142 .sup.a Verapamil 10 113 .sup.b Transwell Studies
Biochanin-A 50 174 .sup.a Indomethacin 100 136 .sup.a
Sequence CWU 1
1
614PRTartificialselenium-dependent thioredoxin reductase active
site 1Gly Cys Xaa Gly 1 24PRTartificialglutaredoxin classical
active site 2Cys Pro Tyr Cys 1 324DNAMus sp.misc_featureprimer
sequence for MDR1 gene 3ctcaccaagc gactccgata catg 24427DNAmus
sp.misc_feature(1)..(27)primer sequence for MDR1 gene 4gataattcct
gtgccaaggt ttgctac 27525DNAmus sp.misc_feature(1)..(25)primer
sequence for MRP-2 gene 5aagacaaaga ttctagtgtt ggacg 25626DNAmus
sp.misc_feature(1)..(26)primer sequence for MRP-2 gene 6agatatgcca
gagatcagtt cacacc 26
* * * * *