U.S. patent application number 09/843796 was filed with the patent office on 2002-03-21 for activating cl- secretion.
This patent application is currently assigned to Southern Research Institute. Invention is credited to Maddry, Joseph A., Sorscher, Eric J..
Application Number | 20020035138 09/843796 |
Document ID | / |
Family ID | 22047784 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020035138 |
Kind Code |
A1 |
Maddry, Joseph A. ; et
al. |
March 21, 2002 |
Activating Cl- secretion
Abstract
Compounds represented by formula (I), 1 wherein A is a 5 or
6-membered unsaturated heterocyclic ring containing at least one N
atom; B is 2 X.sup.2 is O, NH, NR, CH.sub.2, CHR or CR.sub.2, each
R individually is alkyl, cycloalkyl, aryl, alkaryl and aralkyl,
each R.sup.2 individually is H, alkyl, cycloalkyl, aryl, alkaryl,
and aralkyl; Y is a halogen; alkythio group or nitrogenous moiety,
are useful for activating Cl.sup.- secretion, and can be used for
treating cystic fibrosis.
Inventors: |
Maddry, Joseph A.;
(Birmingham, AL) ; Sorscher, Eric J.; (Birmingham,
AL) |
Correspondence
Address: |
Connolly Bove Lodge & Hutz LLP
Suite 800
1990 M Street, N.W.
Washington
DC
20036-3425
US
|
Assignee: |
Southern Research Institute
2000 9th Avenue South
Birmingham
AL
35205
|
Family ID: |
22047784 |
Appl. No.: |
09/843796 |
Filed: |
April 30, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09843796 |
Apr 30, 2001 |
|
|
|
09529915 |
Apr 21, 2000 |
|
|
|
6251930 |
|
|
|
|
09529915 |
Apr 21, 2000 |
|
|
|
PCT/US98/22369 |
Oct 23, 1998 |
|
|
|
60063222 |
Oct 23, 1997 |
|
|
|
Current U.S.
Class: |
514/398 ;
514/384; 514/404 |
Current CPC
Class: |
A61K 31/4164 20130101;
Y10S 514/851 20130101; A61K 31/655 20130101 |
Class at
Publication: |
514/398 ;
514/384; 514/404 |
International
Class: |
A61K 031/4166; A61K
031/4152; A61K 031/4196 |
Claims
What is claimed is:
1. A method for promoting Cl.sup.- secretion in a patient in need
thereof which comprises administering to said patient a composition
comprising a pharmaceutically acceptable carrier and an amount
effective for promoting or activating Cl-- secretion of a compound
represented by the formula: 15wherein A is a 5 or 6 membered
unsaturated heterocyclic ring containing at least one N atom; 16X2
is O, NH, NR, CH.sub.2, CHR or CR.sub.2 each R individually is
alkyl, cycloalkyl, aryl, alkaryl and aralkyl, each R2 individually
is H, alkyl, cycloalkyl, aryl, alkaryl and aralkyl; Y is a halogen;
alkythio group or nitrogenous moiety.
2. The method of claim 1 wherein the alkyl moieties of said R and
R.sup.2 groups contain 1-22 carbon atoms and the aryl moieties of
said R groups contain 6-14 carbon atoms.
3. The method of claim 1 wherein each R individually is selected
from the group consisting of methyl, ethyl, isopropyl, n-butyl,
hexyl, hexenyl, octyl, decyl, dodecyl, phenyl, benzyl and
phenethyl.
4. The method of claim 1 wherein said compound is
5-diazoimidazole-4-carbo- xylic acid n-octyl ester.
5. The method of claim 1 wherein said compound is selected from the
group of compounds represented by the formulae: 17
6. A method for treating a patient suffering from cystic fibrosis
which comprises administering to said patient a composition
comprising a pharmaceutically acceptable carrier and an amount
effective for promoting or activating Cl-- secretion of a compound
represented by the formula: 18wherein A is a 5 or 6 membered
unsaturated heterocyclic ring containing at least one N atom; 19X2
is O, NH, NR, CH.sub.2, CHR or CR.sub.2 each R individually is
alkyl, cycloalkyl, aryl, alkaryl and aralkyl, each R2 individually
is H, alkyl, cycloalkyl, aryl, alkaryl and aralkyl; Y is a halogen;
alkythio group or nitrogenous moiety.
7. The method of claim 6 wherein the alkyl moieties of said R and
R.sup.2 groups contain 1-22 carbon atoms and the aryl moieties of
said R groups contain 6-14 carbon atoms.
8. The method of claim 6 wherein each R individually is selected
from the group consisting of methyl, ethyl, isopropyl, n-butyl,
hexyl, hexenyl, octyl, decyl, dodecyl, phenyl, benzyl and
phenethyl.
9. The method of claim 6 wherein said compound is
5-diazoimidazole-4-carbo- xylic acid n-octyl ester.
10. The method of claim 6 wherein said compound is selected from
the group of compounds represented by the formulae: 20
Description
TECHNICAL FIELD
[0001] The present invention is concerned with activating Cl.sup.-
secretion in a patient. More particularly the present invention is
especially concerned with treating patients suffering from cystic
fibrosis by administering certain heterocyclic nitrogen containing
compounds.
BACKGROUND OF INVENTION
[0002] Insufficient Cl.sup.- transport in epithelial cells has been
associated with disease in individuals and especially with cystic
fibrosis. In particular, the cystic fibrosis transmembrane
conductance regulator (CFTR) functions as a Cl.sup.- channel. At
the surface of epithelial cells. it also regulates the activity of
other ion channels, including amiloride-sensitive Na.sup.+
channels, and other Cl.sup.- transport pathways. Cystic fibrosis
mice lack Cl.sup.- transport capabilities in their nasal airways.
lung cells, and intestines. In humans with cystic fibrosis, sweat
ducts cannot properly reabsorb Cl.sup.-. It has also been suggested
that the Pseudomonas predisposition in cystic fibrosis patients may
be associated with defects in Cl.sup.- reabsorption, rather than
secretion. These results collectively point to defects in Cl.sup.-
transport as responsible for pathogenesis in the disease.
[0003] CFTR regulates airway Na.sup.+ re-absorption through effects
on epithelial sodium channels (ENaCs). CFTR may also regulate
K.sup.+ channels. Several studies have indicated important effects
of wild-type CFTR on membrane turnover in epithelia. including
regulation of both endo- and exocytosis. Acidification of
intracellular compartments such as the golgi and proper in protein
glycosylation and sialation have also been suggested to rely on
normal CFTR function. Although CFTR may subserve other functions in
epithelia, defective Cl.sup.- secretion into the airways is the
best understood and most likely physiologic contributor to clinical
disease. Nasal and lower airway potential difference measurements
indicate abnormal Cl.sup.- secretion into CF airways. Correction of
Cl.sup.- secretory defects are viewed as important and possibly the
benchmark by which to evaluate the effectiveness of therapeutic
interventions such as gene-based or other pharmacologic therapies.
Cystic fibrosis mice are believed to be protected from lung disease
because of alternate Cl.sup.- secretory pathways that are not found
in the intestinal tracts of these animals. accounting for the
absence of lung disease in the animal model. The absence of
alternate Cl.sup.- secretory pathways in murine intestines of mice
may explain the predisposition of CF mice towards lethal
gastrointestinal pathophysiology. Finally, genetic modifiers that
lessen the severity of disease in CF mice appear to activate
alternate Cl.sup.- secretory pathways in the intestines of these
animals. Because of a preponderance of evidence that Cl.sup.-
secretion is not only associated with CF, but directly responsible
for the disease, Cl.sup.- secretagogues form an important aspect of
new pharmacologic approaches to treatment.
[0004] Scientific efforts directed towards identifying activators
of Cl.sup.- secretion in CF tissues are an important aspect of drug
discovery in the disease. Important evidence indicates that drugs
in the sulfonylurea class can maintain certain K.sup.+ channels in
a tonically open-state. Channel openers that might have comparable
action on CFTR or other Cl.sup.- transport pathways are being
actively sought as part of therapeutic development in the disease.
In principal, such agents might either activate residual CFTR
activity at the cell surface, open alternate Cl.sup.- secretory
pathways, or increase the gradient for apical Cl.sup.- secretion
from cells, for example by opening basolateral K.sup.+ channels and
augmenting the tendency for Cl.sup.- to exit at the apical
surface.
[0005] Nucleotides such as uridine triphosphate (UTP) and adenosine
triphosphate (ATP) have been shown to activate Cl.sup.- secretion
in CF tissues, including transepithelial transport in vivo. These
drugs also transiently correct CF bioelectric abnormalities, and
cause strong Cl.sup.- secretion as judged by in vivo measurements
of airway potential difference. However, drugs such as UTP are
rapidly cleared at the airway cell surface by endogenous
ectonucleotidases, and therefore may have limited bioavailablility
in vivo. Moreover, the activities of compounds such as UTP are
often short lived, and disappear within minutes unless fresh
compound is continuously provided on to airway epithelium in vivo.
Tachyphylaxis to UTP has been observed both in vitro and in
vivo.
[0006] The drug CPX, a cyclopropyl xanthene, appears to directly
activate .DELTA.F508 CFTR. CPX exhibits this activity in human CF
pancreatic cells (CFPAC-cell line), in airway cells derived from a
CF patient (IB3-1-cell line), and in NIH 3T3 cells after expression
of the .DELTA.F508 protein. In all cases, Cl.sup.- efflux from
these cells could be activated by CPX. Because CPX is believed to
bind and activate .DELTA.F508 CFTR at the cell surface, the drug
may only be active if sufficient .DELTA.F508 CFTR is present in the
plasma membrane to permit induction of Cl.sup.- transport. Previous
studies have suggested that levels of .DELTA.F508 CFTR present at
the cell surface could be extremely low in vivo due to recognition
of the .DELTA.F508 mutation by protein processing mechanisms in the
endoplasmic reticulum that rapidly degrade the .DELTA.F508
protein.
[0007] In general, Cl.sup.- secretogogues act by elevating
intra-cellular levels of either Ca.sup.2+ or cyclic AMP (cAMP). For
example, agents such as UTP, ATP, NS004, and duramycin are believed
to activate secretion in CF tissues by elevating cellular
Ca.sup.2+. Drugs such as adenosine and milrinone activate residual
CFTR activity by signaling through cAMP and/or PKA.
SUMMARY OF INVENTION
[0008] The present invention is concerned with drugs that exhibit
high activity and long-lived activity for activating Cl.sup.-
secretion from CF airway and pancreatic cells and across cystic
fibrosis tissues and cell monolayers.
[0009] Compounds employed according to the present invention
provide relatively stable activation of Cl.sup.- secretion in CF
cells and tissues and appear to work by a mechanism independent of
either intracellular Ca.sup.2+ or cAMP. Compounds employed
according to the present invention elicit Cl.sup.- secretion that
is active in CF and normal epithelia. and do not appear to require
.DELTA.F508 CFTR at the cell surface.
[0010] The present invention is concerned with promoting or
activating Cl.sup.- secretion in a patient in need thereof by
administering to the patient a composition comprising a
pharmaceutically acceptable carrier and an amount effective for
promoting or activating Cl.sup.- secretion of a compound
represented by the formula: 3
[0011] wherein A is a 5 or 6 membered unsaturated heterocyclic ring
containing at least one N atom; 4
[0012] X.sup.2 is O, NH, NR,
[0013] each R individually is alkyl, cycloalkyl, aryl, alkaryl and
aralkyl,
[0014] each R.sup.2 individually is H, alkyl, cycloalkyl, aryl,
alkaryl and aralkyl;
[0015] Y is a halogen; alkythio group or nitrogenous moiety.
[0016] The present invention is also concerned with treating a
patient suffering from cystic fibrosis by administering to the
patient a composition comprising a pharmaceutically acceptable
carrier and an amount effective for treating cystic fibrosis of a
compound represented by the formula: 5
[0017] wherein A is a 5 or 6 membered unsaturated heterocyclic ring
containing at least one N atom: 6
[0018] X.sup.2 is O, NH, NR,
[0019] each R individually is alkyl, cycloalkyl, aryl, alkaryl and
aralkyl,
[0020] each R.sup.2 individually is H, alkyl, cycloalkyl, aryl,
alkaryl and aralkyl;
[0021] Y is a halogen; alkythio group or nitrogenous moiety.
[0022] Still other objects and advantages of the present invention
will become readily apparent by those skilled in the art from the
following detailed description, wherein it is shown and described
only the preferred embodiments of the invention, simply by way of
illustration of the best mode contemplated of carrying out the
invention. As will be realized the invention is capable of other
and different embodiments, and its several details are capable of
modifications in various obvious respects, without departing from
the invention. Accordingly, the description is to be regarded as
illustrative in nature and not as restrictive.
SUMMARY OF DRAWINGS
[0023] FIGS. 1A-1F are graphs of efflux rate.
[0024] FIGS. 2A-2C are graphs showing efflux rate of iodide in CF
epithelial cells.
[0025] FIGS. 3A-3B show cyclic cAMP and growth characteristics of
CF cells.
[0026] FIGS. 4A-4D illustrate transport effect achieved by the
present invention.
[0027] FIG. 5 illustrates I.sub.Sc response.
[0028] FIGS. 6A-6D show I.sub.Sc response in T84 colonic epithelial
cells.
[0029] FIGS. 7A-7D show I.sub.Sc response in secretory epithelial
cell monolayers.
[0030] FIGS. 8A-8F show Cl.sup.- secretion across polarized
cells.
[0031] FIGS. 9A-9B show transport in CF mouse epithelia.
BEST AND VARIOUS MODES FOR CARRYING OUT INVENTION
[0032] The compounds employed pursuant to the present invention are
represented by the following formula: 7
[0033] wherein A is a 5 or 6 membered unsaturated heterocyclic ring
containing at least one N atom; 8
[0034] X.sup.2 is O, NH, NR,
[0035] each R individually is alkyl, cycloalkyl, aryl, alkaryl and
aralkyl,
[0036] each R.sup.2 individually is H, alkyl, cycloalkyl, aryl,
alkaryl and aralkyl;
[0037] Y is a halogen; alkythio group or nitrogenous moiety.
[0038] Examples of some suitable 5 and 6 membered single ring
groups suitable as the A moiety are 9
[0039] R and R.sup.2 are the same as defined above.
[0040] Examples of some suitable nitrogenous moieties for Y are
N.sub.2, N.sub.3, NO.sub.2, NH.sub.2, CN, NCS, NHR, N(R).sub.2, and
N.dbd.N--N(R).sub.2. R is the same as defined above.
[0041] The alkyl moieties of the R, R.sup.2 and Y groups typically
contain 1-22 carbon atoms and more typically 1-12 carbon atoms. The
cycloalkyl moieties include saturated and unsaturated ring groups
such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl and cyclooctyl. The aryl moieties of the R and R.sup.2
groups typically contain 6 to 14 carbon atoms and 1-3 rings, and
more typically 6 carbon atoms. Examples of suitable R and R.sup.2
groups are methyl, ethyl, isopropyl, n-butyl, isobutyl, hexyl,
hexenyl, octyl, decyl, dodecyl, geranyl, retinyl, phenyl, benzyl
and phenethyl. Although the compounds used in the present invention
have been represented by the above single formula for purposes of
simplicity, it is understood that the electron-rich heterocyclic
base of some of the various compounds is mesoionic and can be
represented by alternative structural formulae. For example, the
preferred compound, 5-diazoimidazale-4-carboxylic acid n-octyl
ester is represented by the following formulae: 10
[0042] Further compounds according to the present invention are
illustrated by the following formulae: 11
[0043] Various of the compounds employed in accordance with the
present invention and method of preparing are described in U.S.
Pat. No. 3,654,257. the entire disclosure of which is incorporated
herein by reference. In addition, Scheme 1 below depicts the
synthetic route that was used for preparing
5-diazoimidazol-4-carboxylic acid n-octyl ester (identified in the
figures as SRI 2931) and can be used to synthesize various of the
disclosed compounds.
[0044] In particular, nitration of commercially available imidazole
1 affords 2, which then undergoes an aldol-type condensation to
give olefin 3. Oxidative cleavage of the double bond, followed by
esterification of the resulting carboxyl moiety with n-octanol, and
subsequent reduction of the nitro substituent, provides amine 6,
which can be converted to the mesoionic
5-diazoimidazol-4-carboxylic acid n octyl ester using standard
diazotization techniques. This same protocol. utilizing alternative
alcohols for the esterification of 4, affords compounds of general
structure 7.
[0045] Scheme 2 below illustrates obtaining compounds having an
azido moiety or cyano moiety. The azido analogs are readily
available from the parent esters or amides by several procedures,
including treatment with hydrazine or thiosemicarbazide. The
corresponding nitrile can be obtained from the diazonium species by
nucleophilic displacement of the diazonium group.
[0046] Scheme 1 below also illustrates the nitro, amino group or
substituted amino at ring position 4. The halogen such as fluoro
groups can be introduced via a Schiemann reaction of the diazonium
species. The isocyanates and isothiocyanates can either be prepared
directly from the diazonium salts, or obtained from the azides via
modified Staudinger or aza-Wittig reactions. The triazenes at
position 4 can be prepared by coupling of the diazoimidazole with
an appropriate primary or secondary amine.
[0047] Removing hydrogen on the ring can be carried out by
substituting the ring nitrogen atom with a lower, non-hydrogen
bonding group such as R.sup.2=methyl. This can be accomplished by
alkylation.
[0048] The amides identified inter alia in Scheme 2 (structure 10)
can be obtained by amination of the corresponding ester. The
ketones (structure 10, X.dbd.CH.sub.2) can be prepared from
intermediates analogous to 3 in Scheme 1. For example.
hydroboration of 3 and its analogs with oxidative work-up
conditions, followed by further oxidation of the resulting carbinol
yield the desired ketones regioselectively.
[0049] Scheme 3 shows preparing a radiolabelled form of
5-diazoimidazol-4-carboxylic acid n-octyl ester. The radiolabelled
compounds are especially useful in evaluating the studying the
mechanism of action of the system. 12 13 14
[0050] The compounds used according to the present invention can be
administered by any means that produces contact of the active
agent's site of action with the desired submucosal gland or surface
epithelium in the body of a human, mammal, bird, or other animal.
They can be administered by any conventional means available for
use in conjunction with pharmaceuticals, either as individual
therapeutic agents or in a combination of therapeutic agents. They
can be administered alone, but generally administered with a
pharmaceutical carrier selected on the basis of the chosen route of
administration and standard pharmaceutical practice.
[0051] The dosage administered will, of course, vary depending upon
known factors, such as the pharmacodynamic characteristics of the
particular agent and its mode and route of administration; the age,
health and weight of the recipient; and the frequency of treatment.
A daily dosage of active ingredient can be expected to provide
about 10 to about 200 .mu.molar to the lower airways, a typical
example being about 100 .mu.molar.
[0052] Dosage forms (compositions suitable for administration)
contain from about 1 mg to about 100 mg of active ingredient per
unit. In these pharmaceutical compositions, the active ingredient
will ordinarily be present in an amount of about 0.5-95% by weight
based on the total weight of the composition.
[0053] The active ingredient can be administered orally in solid
dosage forms, such as capsules, tablets and powders, or in liquid
dosage forms, such as elixirs, syrups and suspensions. It can also
be administered parenterally, in sterile liquid dosage forms. The
active ingredient can also be administered intranasally (nose
drops) or by inhalation. Other dosage forms are potentially
possible such as administration transdermally, via a patch
mechanism or ointment.
[0054] Gelatin capsules contain the active ingredient and powdered
carriers, such as lactose, starch, cellulose, derivatives,
magnesium stearate, stearic acid, and the like. Similar diluents
can be used to make compressed tablets. Both tablets and capsules
can be manufactured as sustained release products to provide for
continuous release of medication over a period of hours. Compressed
tablets can be sugar-coated or film-coated to mask any unpleasant
taste and protect the tablet from the atmosphere, or enteric coated
for selective disintegration in the gastrointestinal tract.
[0055] Liquid dosage forms for oral administration can contain
coloring and flavoring to increase patient acceptance.
[0056] In general, water, a suitable oil, saline, aqueous dextrose
(glucose) and related sugar solutions and glycols, such as
propylene glycol or polyethylene glycols are suitable carriers for
parenteral solutions. Solutions for parenteral administration
preferably contain a water-soluble salt of the active ingredient,
suitable stabilizing agents, and if necessary, buffer substances.
Antioxidizing agents such as sodium bisulfite, sodium sulfite or
ascorbic acid, either alone or combined, are suitable stabilizing
agents. Also used are citric acid and its salts and sodium EDTA. In
addition, parenteral solutions can contain preservatives, such as
benzalkonium chloride, methyl- or propylparaben, and
chlorobutanol.
[0057] Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, Mack Publishing Company, a
standard reference text in this field.
[0058] Useful pharmaceutical dosage forms for administration of the
compounds of this invention can be illustrated as follows:
[0059] Capsules
[0060] A large number of unit capsules are prepared by filling
standard two-piece hard gelatin capsules each with 100 mg of
powdered active ingredient, 150 mg of lactose, 50 mg of cellulose,
and 6 mg of magnesium stearate.
[0061] Soft Gelatin Capsules
[0062] A mixture of active ingredient in a digestible oil such as
soybean oil, cottonseed oil, or olive oil is prepared and injected
by means of a positive displacement pump into gelatin to form soft
gelatin capsules containing 100 mg of the.active ingredient. The
capsules are washed and dried.
[0063] Tablets
[0064] A large number of tablets are prepared by conventional
procedures so that the dosage unit was 100 mg of active ingredient,
0.2 mg of colloidal silicon dioxide, 5 mg of magnesium stearate,
275 mg of microcrystalline cellulose, 11 mg of starch, and 98.8 mg
of lactose. Appropriate coatings may be applied to increase
palatability or delay absorption.
[0065] Moreover, the compounds of the present invention can be
administered in the form of nose drops or a nasal inhaler.
[0066] Various modifications of the invention in addition to those
shown and described herein will be apparent to those skilled in the
art from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims.
[0067] The foregoing disclosure includes all the information deemed
essential to enable those skilled in the art to practice the
claimed invention. Because the cited applications may provide
further useful information, these cited materials are hereby
incorporated by reference in their entirety.
[0068] In tissue monolayers, the drug leads to stable activation of
Cl.sup.- secretion that is well maintained even following removal
of drug from the solution bathing the cells. This compound's
activity is observed specifically after administration to the
apical, and not the basolateral, surface of polarized airway,
pancreatic, and colonic epithelial cells.
[0069] Test for Activation of Anion Efflux from Cystic Fibrosis
Cells
[0070] The iodide efflux protocol is a conventional test for the
existence of anion permeability pathways, including both CFTR
(cAMP-activated) and non-CFTR permeability pathways (e.g.,
Ca.sup.2+-activated Cl.sup.- transport). FIG. 1 establishes that
5-diazoimidazol-4-carboxylic acid n-octyl ester (so-called compound
"SRI 2931") confers a rapid efflux of iodide in CF epithelial cells
similar to that observed in cells containing wild-type CFTR and
stimulated by action of cAMP (i.e., T84 colonic carcinoma cells
treated with 10 .mu.M forskolin, data not shown). In addition, this
effect of 5-diazoimidazol-4-carboxylic acid n-octyl ester does not
appear to be Ca.sup.2+-dependent, since removal of extracellular
Ca.sup.2+ (a maneuver which depletes both intra- and extracellular
Ca.sup.2+ stores) does not substantially inhibit anion efflux.
Ionomycin, a Ca.sup.2+ ionophore, is known to activate
non-CFTR-type anion efflux pathways in CFPAC cells by increasing
intracellular Ca.sup.2+. Ionomycin-mediated anion transport in
CFPAC cells was studied and this control indicated the anticipated
stimulation of iodide efflux by ionomycin.
[0071] FIG. 2. Anion efflux protocol in human epithelial cells.
[0072] Epithelial cells in the pancreatic ducts, lung, and colon
secrete fluid and electrolytes into the pancreatic ductular lumen,
airways and intestinal lumen, respectively. The inability to
secrete anions in response to a cAMP-dependent agonist such as
forskolin is a hallmark of the CF phenotype. Note that epithelial
cells derived from CF pancreatic ductular cells (FIG. 1), or
primary airway cells taken from a CF patient (FIG. 2), lack
forskolin-activated anion permeability pathways, since they lack
CFTR. Normal human colonic epithelial cells (T84 cells, which
express high levels of CFTR) exhibit strong activation of anion
efflux due to forskolin (not shown). Iodide efflux is strongly
activated in all three cell types by 5-diazoimidazol-4-carboxylic
acid n-octyl ester, indicating that these effects are not dependent
on the presence of wild-type CFTR. Methods: Iodide efflux. Iodide
efflux was performed as previously described (Venglarik et al., Am.
J. Physiol. 259:C358-C364, 1990, and Drumm et al., Cell
62:1227-1233, 1990) Cells were grown on 35 mM culture dishes, and
effluxes were performed when the cells were 80-100% confluent using
a phosphate buffered Ringer's solution. Cells were loaded with
.sup.125I (2-5 .mu.Ci/ml) for 30 minutes and then washed with PBS
to remove extracellular .sup.125I. Efflux was detected by measuring
the radioactivity in the extracellular solution which was changed
every 15 seconds. At specific points, compounds were added to the
cells. Increases in the rate of efflux after addition of a compound
indicates the activation of an anion permeability pathway.
[0073] FIG. 3. 5-diazoimidazol-4-carboxylic acid n-octyl ester does
not increase cellular cAMP and allows proliferation of cells at
concentrations that activate anion efflux. (3A) Cell proliferation
assay in the presence and absence of 5-diazoimidazol-4-carboxylic
acid n-octyl ester.
[0074] Proliferation was measured as in Hughes et al., Tumor
specific killing with high bystander toxicity using the human
tyrosinase promoter to express the E. coli PNP gene, Cancer
Research 55: 3339-3345, 1995 and Parker et al., In vivo gene
therapy of cancer with E. coli purine nucleoside phosphorylase,
Human Gene Therapy, 1997 (in press) using a commercially available
kit (Cell titer 96 kit, Promega) and carried out according to
manufacturer's protocol. 20 .mu.m 2931 activates Cl.sup.- secretion
in CF cells within 5 minutes. Even when these cells are grown for 7
days in 2931 at 20 .mu.m, minimal effects on cellular proliferation
are observed, and an initial growth delay is overcome by day 7 of
the experiment. (FIG. 3A) cAMP levels were tested using the cAMP
Immunoassay kit (Cayman) according to manufacturer's protocol.
[0075] These results demonstrate 1) the tested diazoimidazole
causes anion release from cells lacking functional CFTR, 2) the
component works in both human CF pancreatic cells and human airway
cells, 3) the drug is active in both a cell line and in primary
cells, and 4) the new drug has activity on single cell transport,
establishing that it can mediate effects by augmenting anion
transport at the plasma membrane, so that transcellular (as opposed
to paracellular) effects on Cl.sup.- transport across a cell
monolayer or tissue can also be predicted.
[0076] FIG. 3 shows that under the same conditions than anion
transport is stimulated, no increase in cellular cAMP levels within
cells can be measured (FIG. 3B), and CFPAC cells continue to
proliferate even after several days of exposure to a drug that
activates Cl.sup.- secretion within minutes (FIG. 3A). Taken
together, this data indicates that the effect of the compounds
employed according to the present invention is unlikely to be
cAMP-mediated and does not appear toxic to cells at the
concentrations studied. In contrast to drugs such as ionomycin, the
transient effects do not appear strongly dependent on extracellular
Ca.sup.2+, so it is unlikely that the compounds employed in the
present invention act as a Ca.sup.2+ ionophore or, for example,
through non-specific effects on the cell membrane that would allow
Ca.sup.2+ influx. The activity is also different from drugs
believed to act through cAMP-protein Kinase A such as milronone or
adenosine.
[0077] Test for Activation of Transepithelial Chloride Transport in
CF Epithelial Cell Monolayers
[0078] FIG 4. Cl.sup.- secretion in CFPAC-1 cells. CFPAC-1 cell
monolayers were grown to confluency on permeable supports
(Millipore) for 10 days. Filters were mounted in an Ussing chamber,
and short circuit current measurement was carried out as previously
described (Venglarik and Dawson, Am. J. Physiol. 251:C563-C570,
1986). Filters were bathed in Ringer's solution (in mM: 145
Na.sup.+, 5 K.sup.+, 124.8 Cl.sup.-, 1.2 Ca.sup.2+, 1.2 Mg.sup.2+,
25 HCO.sub.3--, 4.2 PO.sub.4, 10 glucose; pH=7.4) on the serosal
surface and in a 6 mM Cl.sup.- Ringer's solution (118.8 mM of
Cl.sup.- was replaced by the impermeant anion gluconate) on the
mucosal surface. The compounds were screened using different
concentrations of drugs and adding them to alternate surfaces of
the filters (mucosal; serosal; mucosal and serosal). The effects of
forskolin (10 .mu.M), UTP (100 .mu.M) CPX (100 .mu.m) and duramycin
(5 .mu.m) were also studied.
[0079] In order to test 5-diazoimidazol-4-carboxylic acid n-octyl
ester in transepithelial Cl.sup.- transport, CFPAC cells were crown
as a low resistance monolaver on permeable supports. Explants of CF
nasal polyps (primary airway epithelial cells) and control, non-CF
colonic epithelial cells (T84) containing wild-type CFTR were also
studied. In the presence of a serosal-to-mucosal Cl.sup.- gradient,
addition of the drug to the apical (but not basolateral) surface
led to a strong I.sub.SC response in the direction of Cl.sup.-
secretion (FIG. 4A). The I.sub.SC is Cl.sup.--dependent and was
much less pronounced when Cl.sup.- was omitted from the mucosal and
serosal bathing solutions (FIG. 4B). The direction of I.sub.SC
could be reversed by reversing the Cl.sup.- gradient (i.e., from
mucosal-to-serosal), supporting the notion that Cl.sup.- transport
was responsible for the I.sub.SC caused by the
5-diazoimidazol-4-carboxyl- ic acid n-octyl esters (FIG. 4D).
Similar I.sub.SC activation by this compound was also observed in
cystic fibrosis primary airway epithelial cells (FIG. 5), and in
T84 colonic epithelial cells (FIG. 6) grown as monolayers on
permeable supports. The 5-diazoimidazol-4-carboxylic acid n-octyl
ester augmented the maximal I.sub.SC activation caused by 10 .mu.M
forskolin, suggesting that it works through a mechanism different
from cAMP activation and that the drug further augments the
I.sub.SC present in (secretory) epithelial cell monolayers due to
increased cAMP (FIG. 6). In other words, these experiments raise
the possibility that Cl.sup.- secretion effects of drugs such as
adenosine, milrinone, IBMX, or forskolin might be increased by
compounds employed in the present invention by a conjoint therapy
treatment. Amiloride (10 .mu.M) applied to the apical surface of
CFPAC cells did not alter this response (suggesting that the effect
is not mediated by Na.sup.+ reabsorption; note that CFPAC cells do
not normally perform Na.sup.+ reabsorption as they are derived from
a cell type (pancreatic ductular cells) that are not Na.sup.+
reabsorptive). BAPTA AM (100 .mu.M, to deplete intracellular
Ca.sup.2+, and thapsagargin (0.1 .mu.M, to prevent release of
intracellular Ca.sup.2+ stores) also had no effect on the
5-diazoimidazol-4-carboxylic acid n-octyl ester-induced I.sub.SC
(FIG. 7). These results indicate that the compound acts through a
mechanism different from previous modes of activating Cl.sup.-
secretion in CF cells, such as cyclic AMP or Ca.sup.+2. The
I.sub.SC was not likely to be caused by HCO.sub.3-secretion, since
an inhibitor of HCO.sub.3-production (acetazolimide, 100 .mu.M) did
not inhibit the effect of the diazoimidazol. The activation of
I.sub.SC also does not appear to require an imposed gradient across
the cell monolayer. In symmetrical Ringer's lactate (equivalent
NaCl on both sides of the monolayer), activation of I.sub.SC in the
direction of Cl.sup.- secretion was still present, indicating that
cells remain active and able to maintain a gradient for Cl.sup.-
secretion after addition of the 5-diazoimidazol-4-carboxylic acid
n-octyl ester. The I.sub.SC occurs after mucosal, but not serosal
addition of the compound.
[0080] Simplified Model of Epithelial Cl.sup.- Secretion
[0081] Epithelial cells accumulate Cl.sup.- and K.sup.+
intracellularly by virtue of coordinated activity of a Na/K/2Cl
co-transporter and the Na/K-ATPase in the basolateral membranes of
Cl.sup.- secreting epithelia. A strong electrochemical driving
force (for the exit of K.sup.+ ions through basolateral K.sup.+
channels and the exit of Cl.sup.- ions through apical Cl.sup.-
channels) exists in these cells in their resting state. Increases
in cellular cAMP open both K.sup.+ and Cl.sup.- channels in normal
tissues, resulting in fluid and electrolyte (Cl.sup.-) secretion
across the apical membrane and into the lumen. It is believed that
the 5-diazoimidazol-4-carboxylic acid n-octyl ester works by
directly augmenting the permeability of the apical membrane of
polarized epithelial cells to ions such as Cl.sup.-. In particular,
it augments anion permeability in lung, pancreas, and colonic
epithelium by an anion efflux protocol (FIGS. 1 and 2). Second, the
compound acts only at the apical, but not the basolateral cell
surface in these cells (FIG. 4). Third, it does not appear to act
through cell signalling pathways such as Ca.sup.2+ or cAMP that are
known to open epithelial Cl.sup.- channels. Fourth, the effects are
additive with another agent (forskolin) that raises cellular cAMP
and increases the driving force across the apical membrane for
Cl.sup.- secretion. Each of these observations supports the belief
that 5-diazoimidazol-4-carboxylic acid n-octyl ester directly and
specifically activates Cl.sup.- permeability in the apical
membranes of human epithelial cells derived from many tissues.
[0082] The activity of 5-diazoimidazol-4-carboxylic acid n-octyl
ester was of longer duration than other drugs (UTP, CPX, or
duramycin) studied under the same conditions in CFPAC cells (FIG.
8). CPX of (100 .mu.M) had an effect on transepithelial transport
that did not suggest strong Cl.sup.- secretion, even in the
presence of a serosal-mucosal Cl.sup.- gradient. Although the
concentration of CPX used here was the same as that reported to
activate Cl.sup.- efflux for CFPAC cells, the predominant I.sub.SC
was in a direction opposite to that expected for Cl.sup.-
secretion. UTP had a small, transient activation I.sub.SC in CFPAC
cells, and duramycin, a drug believed to act through increased
intracellular Ca.sup.2+ confirmed a stronger, although transient,
activation of I.sub.sc in the same system, again in a direction
compatible with Cl.sup.- secretion. UTP and CPX have both been used
as part of clinical trials as Cl.sup.- secretagogues in CF.
5-diazoimidazol-4-carboxylic acid n-octyl ester has more potent and
prolonged effects than either of these drugs, is believed to work
by a different mechanism, and is therefore expected to have
substantial advantages over any available compound for activating
Cl.sup.- secretions in CF tissues.
[0083] These results confirm activity of a Cl.sup.--dependent
I.sub.SC in CF epithelial cells treated with
5-diazoimidazol-4-carboxylic acid n-octyl ester. Drugs such as
forskolin and CPX do not mediate Cl.sup.- secretion in CF cells by
this protocol, and UTP mediates a smaller, transient effect.
Because the I.sub.SC could be reversed by switching the Cl.sup.-
concentration gradient, part of the effect could be paracellular.
However, the direct effects of 5-diazoimidazol-4-carboxylic acid
n-octyl ester on anion efflux in single cells independent of
cellular junctions (FIG. 2) has been demonstrated in our
experiments. Moreover, the I.sub.SC activation is also noted when
symmetrical bathing solutions are present, indicating that an
important part of the effect is due to a transcellular pathway.
These tests establish that the compounds employed in this invention
act as strong stimulators of anion transport in single CF cells and
CF cell monolayers in vitro.
[0084] C.4. Studies of Diazoimidazole and Chloride Secretion in
Intact Tissues
[0085] 5-diazoimidazol-4-carboxylic acid n-octyl ester activates
I.sub.SC across a mouse intestine when added to the mucosal
surface. The activation is similar to that observed for primary CF
airway cells and CFPAC cell monolayers, and occurs in normal mice
(A) and in .DELTA.F508 CFTR transgenic mice (B) (FIG. 9). I.sub.SC
activation is also observed in CFTR knock out (-/-) mice (data not
shown), indicating that the action elicited by compound does not
require expression of either wild-type or mutant CFTR protein.
[0086] FIG. 9. Transport in CF Mouse Intestinal Epithelia
[0087] Small sections of mouse colon from both wild-type and CF
knockout mice were mounted in Ussing chambers, and I.sub.SC
measurements were recorded as described above. Both mucosal and
serosal surfaces were bathed in regular Ringer's solution, and TTX
(1.54.times.10.sup.-3 .mu.M) was added to the serosal side of the
tissue. After a steady-state had been obtained, amiloride
(10.sup.-4 M) was added to the mucosal surface. Both sides were
then bathed with regular Ringer's. A Cl gradient was established by
bathing the mucosal surface with a 6 mM Cl.sup.- solution.
5-diazoimidazol-4-carboxylic acid n-octyl ester (100 .mu.M) was
added first to the mucosal and then the serosal surface of the
mounted tissue (FIG. 9).
[0088] The foregoing description of the invention illustrates and
describes the present invention. Additionally, the disclosure shows
and describes only the preferred embodiments of the invention but,
as mentioned above, it is to be understood that the invention is
capable of use in various other combinations, modifications, and
environments and is capable of changes or modifications within the
scope of the inventive concept as expressed herein, commensurate
with the above teachings and/or the skill or knowledge of the
relevant art. The embodiments described hereinabove are further
intended to explain best modes known of practicing the invention
and to enable others skilled in the art to utilize the invention in
such, or other, embodiments and with the various modifications
required by the particular applications or uses of the invention.
Accordingly, the description is not intended to limit the invention
to the form disclosed herein. Also, it is intended that the
appended claims be construed to include alternative
embodiments.
* * * * *