U.S. patent application number 14/829234 was filed with the patent office on 2016-01-07 for compounds as modulators of a mutant cftr protein and their use for treating diseases associated with cftr protein malfunction.
The applicant listed for this patent is INSTYTUT BIOCHEMII BIOFIZYKI PAN. Invention is credited to Aleksander EDELMAN, Janine FRITSCH, Norbert ODOLCZYK, Danielle TONDELIER, Grzegorz WIECZOREK, Piotr ZIELENKIEWICZ.
Application Number | 20160000778 14/829234 |
Document ID | / |
Family ID | 44356169 |
Filed Date | 2016-01-07 |
United States Patent
Application |
20160000778 |
Kind Code |
A1 |
ODOLCZYK; Norbert ; et
al. |
January 7, 2016 |
COMPOUNDS AS MODULATORS OF A MUTANT CFTR PROTEIN AND THEIR USE FOR
TREATING DISEASES ASSOCIATED WITH CFTR PROTEIN MALFUNCTION
Abstract
An exemplary embodiment relates to novel protein modulators
capable of altering function of the mutant CFTR protein and their
use for treating diseases associated with CFTR protein malfunction.
An exemplary embodiment provides compositions, pharmaceutical
preparations and methods of correcting the cellular alteration of a
mutant CFTR protein wherein the CFTR mutation is a mutation
.DELTA.F508-CFTR, or another mutation of class II.
Inventors: |
ODOLCZYK; Norbert;
(Warszawa, PL) ; ZIELENKIEWICZ; Piotr; (Warszawa,
PL) ; WIECZOREK; Grzegorz; (Warszawa, PL) ;
EDELMAN; Aleksander; (Chatenay-Malabry, FR) ;
TONDELIER; Danielle; (Le Mesnil-le-Roi, FR) ;
FRITSCH; Janine; (Saint Michel sur Orge, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTYTUT BIOCHEMII BIOFIZYKI PAN |
Warszawa |
|
PL |
|
|
Family ID: |
44356169 |
Appl. No.: |
14/829234 |
Filed: |
August 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13822584 |
Apr 4, 2013 |
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PCT/US2011/000060 |
Jun 20, 2011 |
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14829234 |
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Current U.S.
Class: |
514/253.03 ;
544/361 |
Current CPC
Class: |
A61K 31/496 20130101;
A61K 31/52 20130101; A61P 9/00 20180101; A61K 31/663 20130101; A61P
13/00 20180101; A61P 1/00 20180101; A61P 43/00 20180101; A61P 1/18
20180101; A61P 15/00 20180101; C07C 2603/18 20170501; C07D 401/12
20130101; C07C 235/84 20130101; C07D 473/22 20130101; C07D 473/30
20130101; A61P 11/00 20180101; C07F 9/305 20130101; C07D 219/10
20130101; A61P 19/04 20180101; A61K 31/194 20130101; C07F 9/304
20130101; C07C 233/65 20130101 |
International
Class: |
A61K 31/496 20060101
A61K031/496 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 14, 2010 |
PL |
P392 396 |
Sep 14, 2010 |
PL |
P392 397 |
Claims
1. A composition comprising: a modulator of a mutant CFTR protein
adapted for use in the manufacture of a medication for the
treatment of diseases associated with CFTR protein malfunction
including: a compound of general formula (I) ##STR00018## its
tautomers, E and Z geometrical isomers, optically active forms such
as enantiomers, diastereomers and their racemate forms or a mixture
of stereoisomeric forms or its pharmaceutically acceptable salts
thereof or complexes thereof; wherein Z.sup.1 is independently
selected from the group consisting of: --C.sub.nH.sub.(2n)--, which
is branched or unbranched wherein n is an integer from 1 to 5;
--C.sub.nH.sub.(2n-2)-- in E or Z geometrical conformation which is
branched or unbranched wherein n is an integer from 2 to 5;
--C.sub.nH.sub.(2n-4)-- which is branched or unbranched wherein n
is an integer from 2 to 5; --CR'H--, --C.sub.2H.sub.3R'--, E or
Z--C.sub.2HR'--, --C.sub.3H.sub.5R'--, E or Z--C.sub.3H.sub.3R'--,
--OCH.sub.2--, --CH.sub.2O--, --NR''CH.sub.2--, --CH.sub.2NR''--;
wherein R' is independently selected from the group consisting of:
--H, halogen, --NH.sub.2, --OH, --CN, CF.sub.3, --CHF.sub.2,
--CH.sub.2F, --SH, --SCN, --CH.sub.3, --C.sub.2H.sub.5; wherein R''
is independently selected from the group consisting of: --H,
--CH.sub.3, --C.sub.2H.sub.5; wherein R.sup.1 and R.sup.2 are
independently selected from the group consisting of aromatic ring
or heteroaromatic ring.
2. The composition of claim 1, wherein R.sup.1 and R.sup.2 are
independently selected from the group of sub-formula (Ia):
##STR00019## wherein A.sub.1, A.sub.2, A.sub.3, A.sub.4, A.sub.5,
A.sub.6 is independently selected N or C atoms wherein ring contain
0-3 nitrogen atoms; wherein E.sup.1, E.sup.2, E.sup.3, E.sup.4,
E.sup.5 represents optional substituents, which are selected from:
--OR.sub.B, --OC(.dbd.O)R.sub.C, --OC(.dbd.O)OR.sub.B,
--OC(.dbd.O)N(R.sub.A)R.sub.A', --C(.dbd.O)R.sub.C,
--C(.dbd.O)N(R.sub.A)R.sub.A', --C(.dbd.O)N(OR.sub.B)R.sub.A,
--C(.dbd.O)OR.sub.B, --C(.dbd.S)R.sub.C,
--C(.dbd.O)C(.dbd.O)R.sub.C, --CH.sub.2OR.sub.B,
--CH.sub.2CH.sub.2OR.sub.B, --CH.sub.2N(R.sub.A)R.sub.A',
--CH.sub.2CH.sub.2N(R.sub.A)R.sub.A', --CH.sub.2OCH.sub.2R.sub.C,
--CH.sub.2N(R.sub.A)CH.sub.2R.sub.C, --SR.sub.D,
--S(.dbd.O)R.sub.D, --SO.sub.2R.sub.D,
--SO.sub.2N(R.sub.A)R.sub.A', --SO.sub.3R.sub.B,
--N(R.sub.A)C(.dbd.O)R.sub.C, --N(R.sub.A)C(.dbd.O)OR.sub.B,
--N(R.sub.A)C(.dbd.O)N(R.sub.A')R.sub.A'',
--N(R.sub.A)SO.sub.2R.sub.D,
--N(R.sub.A)SO.sub.2N(R.sub.A')R.sub.A'', --N(R.sub.A)R.sub.A',
--N(R.sub.A)C(.dbd.O)R.sub.C, --N(R.sub.A)C(.dbd.O)OR.sub.B,
--N(R.sub.A)N(R.sub.A')R.sub.A'',
--N(R.sub.A')N(R.sub.A)C(.dbd.O)R.sub.C, --NO.sub.2, --CN,
--CF.sub.3, --CHF.sub.2, --CH.sub.2F, --NH.sub.2, --SCN,
--SO.sub.2CN, --F, Cl, --Br, --I, --PO.sub.3H.sub.2,
--OPO.sub.3H.sub.2, --C.sub.nH.sub.2nR.sub.C which is branched or
unbranched wherein n is an integer from 1 to 5;
--C.sub.nH.sub.(2n-2)R.sub.C in E or Z geometrical conformation
which branched or unbranched wherein n is an integer from 2 to 5;
--C.sub.nH.sub.(2n-4)R.sub.C which is branched or unbranched
wherein n is an integer from 2 to 5; wherein R.sub.A, R.sub.A',
R.sub.A'' are each independently selected from the group consisting
of: --H, lower alkyl group, --CN, --CF.sub.3, --CHF.sub.2,
--CH.sub.2F, --OH; wherein R.sub.B is independently selected from
the group consisting of: --H, lower alkyl group, --CN, --CF.sub.3,
--CHF.sub.2, --CH.sub.2F, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2I;
wherein R.sub.C is independently selected from the group consisting
of: --H, lower alkyl group, --CN, --CF.sub.3, --CHF.sub.2,
--CH.sub.2F, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2I, --F, --Cl,
--Br, --I, --NH.sub.2, wherein R.sub.D is independently selected
from the group consisting of: --H, lower alkyl group.
3. The composition of claim 1, wherein the compound is represented
by the following structures: ##STR00020##
4. The composition of claim 2, wherein the compound is represented
by the following structures: ##STR00021##
5. The composition of claim 1, wherein the compound has an effect
on mutant CFTR protein, wherein the mutant CFTR protein is a
mutation .DELTA.F508-CFTR, or another mutation of class II, and
wherein a mutation .DELTA.F508-CFTR, or another mutation of class
II are involved in CFTR protein malfunction.
6. The composition of claim 2, wherein the compound has an effect
on mutant CFTR protein, wherein the mutant CFTR protein is a
mutation .DELTA.F508-CFTR, or another mutation of class II, and
wherein a mutation .DELTA.F508-CFTR, or another mutation of class
II are involved in CFTR protein malfunction.
7. The composition of claim 3, wherein the compound has an effect
on mutant CFTR protein, wherein the mutant CFTR protein is a
mutation .DELTA.F508-CFTR, or another mutation of class II, and
wherein a mutation .DELTA.F508-CFTR, or another mutation of class
II are involved in CFTR protein malfunction.
8. The composition of claim 4, wherein the compound has an effect
on mutant CFTR protein, wherein the mutant CFTR protein is a
mutation .DELTA.F508-CFTR, or another mutation of class II, and
wherein a mutation .DELTA.F508-CFTR, or another mutation of class
II are involved in CFTR protein malfunction.
9. The composition of claim 5, wherein the disease associated with
CFTR protein malfunction is cystic fibrosis.
10. The composition of claim 6, wherein the disease associated with
CFTR protein malfunction is cystic fibrosis.
11. The composition of claim 7, wherein the disease associated with
CFTR protein malfunction is cystic fibrosis.
12. The composition of claim 8, wherein the disease associated with
CFTR protein malfunction is cystic fibrosis.
13. The compound of claim 1, wherein said compound is a modulator
for use in the treatment of cystic fibrosis wherein it has effect
on CFTR-dependent ion transport across cellular membrane and/or it
has the ability to increase the number of mutant CFTR proteins that
reach the cell surface.
14. The compound of claim 1, wherein said compound is a modulator
for use in the treatment of cystic fibrosis wherein it has
stabilizing effect on the structure of the mutant CFTR protein
and/or blocks the interaction with cellular proteins responsible
for the premature degradation of mutant CFTR.
15. The compound of claim 1, wherein said compound is a modulator
for use in the treatment of cystic fibrosis wherein it has effect
on mutant CFTR protein, wherein said CFTR mutation is a mutation
.DELTA.F508-CFTR, or another mutation of class II.
16. A method comprising: treating cystic fibrosis in a patient
including administering a therapeutically effective amount of a
compound of a general formula (I), as a modulator of a mutant CFTR
protein to the patient having cystic fibrosis: ##STR00022## its
tautomers, E and Z geometrical isomers, optically active forms such
as enantiomers, diastereomers and their racemate forms or a mixture
of stereoisomeric forms or its pharmaceutically acceptable salts
thereof or complexes thereof; wherein Z.sup.1 is independently
selected from the group consisting of: --C.sub.nH.sub.(2n)--, which
is branched or unbranched wherein n is an integer from 1 to 5;
--C.sub.nH.sub.(2n-2)-- in E or Z geometrical conformation which is
branched or unbranched wherein n is an integer from 2 to 5;
--C.sub.nH.sub.(2n-4)-- which is branched or unbranched wherein n
is an integer from 2 to 5; --CR'H--, --C.sub.2H.sub.3R'--, E or
Z--C.sub.2HR'--, --C.sub.3H.sub.5R'--, E or Z--C.sub.3H.sub.3R'--,
--OCH.sub.2--, --CH.sub.2O--, --NR''CH.sub.2--, --CH.sub.2NR''--;
wherein R' is independently selected from the group consisting of:
--H, halogen, --NH.sub.2, --OH, --CN, CF.sub.3, --CHF.sub.2,
--CH.sub.2F, --SH, --SCN, --CH.sub.3, --C.sub.2H.sub.5; wherein R''
is independently selected from the group consisting of: --H,
--CH.sub.3, --C.sub.2H.sub.5; wherein R.sup.1 and R.sup.2 are
independently selected from the group consisting of aromatic ring
or heteroaromatic ring and wherein the compound of effects
CFTR-dependent ion transport across a cellular membrane and/or the
compound increases the number of mutant CFTR proteins reaching the
cell surface.
17. The method according to claim 16 wherein the compound has a
stabilizing effect on the structure of the mutant CFTR protein
and/or blocks the interaction with cellular proteins responsible
for the premature degradation of mutant CFTR.
18. The method according to claim 16 wherein the compound is
effecting a mutant CFTR protein, wherein said CFTR mutation is a
mutation .DELTA.F508-CFTR, or another mutation of class II.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This divisional application claims benefit of U.S.
application Ser. No. 13/822,584 filed Mar. 12, 2013, the disclosure
of which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The exemplary techniques disclosed herein relate to novel
protein modulators capable of altering function of the mutant CFTR
protein and their use for treating diseases associated with CFTR
protein malfunction. Exemplary embodiments provide compositions,
pharmaceutical preparations and methods of correcting the cellular
alteration of a mutant CFTR protein wherein the CFTR mutation is a
mutation .DELTA.F508-CFTR, or another mutation of class II.
BACKGROUND
[0003] Cystic fibrosis (also known as CF or mucoviscidosis) is one
of the most common, fatal genetic diseases in humans. CF is an
inherited autosomal recessive genetic disease that affects around 1
child in 2,500 live births. CF is caused by mutations in the cftr
gene that encodes the cystic fibrosis transmembrane conductance
regulator (CFTR protein) with activity as an epithelial chloride
ion channel. As a result of impaired function of this protein,
severe symptoms associated with respiratory and digestive systems
and male reproductive system appear. To date, more than 1600
mutations in CFTR gene have been identified and described.
[0004] The CFTR gene mutations were classified into five classes
based on the molecular mechanisms leading to the CFTR protein
malfunction. The class I mutations contribute to the formation of
proteins with incomplete length and usually involve the complete
loss of its activity (e.g. G542X). Mutation in the class II lead to
abnormal maturation of proteins in the endoplasmic reticulum and
Golgi apparatus. The effect of these mutations is premature
degradation of the protein. Hence, CFTR does not reach the cell
membrane where it should perform its function (eg, .DELTA.F508,
.DELTA.I507, S549R). The gene product having mutations of class III
is properly synthesized, transported and incorporated into the cell
membrane, but has decreased activity caused by abnormal regulation
of the protein.
[0005] These mutations are frequently situated within one of the
nucleotide binding domain. (eg. G551D/S). Mutations of class IV
cause anomalies in the structure of the transmembrane protein and
thereby reduce the conduction of chloride channel (e.g. R117H,
R334W). Mutations altering the stability of mRNA represent a class
V of the mutations of the CFTR gene (3849+10kbC->T, 5T).
[0006] The most prevalent mutation present in at least one allele
in approximately 90% of patients is a deletion of phenylalanine at
position 508 of the CFTR amino acid sequence (.DELTA.F508 CFTR).
This is a classic example of class II mutation that causes
premature degradation of the protein. This mutation is associated
with water-electrolyte disturbances (among others with chloride
anion flux out of a cell across the plasma membrane and the
movement of sodium ions into the cell) and results in the
appearance of pathological symptoms. Some of the most severe
symptoms include congestion and increased mucus viscosity in the
upper and lower airways leading to lung damage. These conditions
create a favorable environment for development of bacterial
infections caused by e.g. Pseudomonas aeruginosa. Moreover,
malfunction of CFTR protein leads to obstruction of exocrine
pancreatic ducts and related digestive disorders.
[0007] CFTR is a glycoprotein with 1480 amino acids and classified
as an ABC (ATP-binding cassette) transporter. The protein consists
of five domains. There are two nucleotide binding domains (NBD1 and
NBD2), regulatory domain (RD) and two transmembrane domains (TMD1
and TMD2). The protein activity is regulated by cAMP-dependent
Protein Kinase (PKA) which catalyze phosphorylation of regulatory
domain (RD) and also by binding of two ATP molecules to NBD1 and
NBD2 domains.
[0008] The disclosure in the patent application WO2007075901 (publ.
2007-Jul.-5) relates to prodrugs of modulators of ABC transporters,
particularly, CFTR modulators, compositions thereof, and methods
therewith. An exemplary embodiment also relates to methods of
treating ABC transporter mediated diseases using such
modulators.
[0009] In U.S. Patent Publication No. 20080319008, compounds that
increase activity (ion transport) of a mutant CFTR protein, and
uses thereof are described. The disclosure also provides
compositions, pharmaceutical preparations and methods increasing
ion transport activity of a mutant CFTR protein, i.e. .DELTA.F508
CFTR, G551D-CFTR, G1349D-CFTR or D1152H-CFTR, that are suitable in
treating cystic fibrosis (CF). The compositions and pharmaceutical
preparations of the disclosure may comprise one or more
phenylglycine-containing compounds or sulfonamide-containing
compounds or an analog or derivatives thereof.
[0010] In a publication WO2009051910, compounds that increase ion
transport activity of a mutant CFTR protein, and uses thereof are
described. The disclosure provides compositions, pharmaceutical
preparations and methods for increasing activity of a mutant-CFTR.
The compositions, pharmaceutical preparations and methods are
notable for the study and treatment of disorders associated with
mutant-CFTR, such as cystic fibrosis. The compositions and
pharmaceutical preparations of the disclosure may comprise one or
more phenylglycine-containing compounds, or an analog or derivative
thereof.
[0011] U.S. Pat. No. 5,948,814 describes the use of genistein
compound for treatment of CF. A method of treating cystic fibrosis
by generating CFTR function in cells containing mutant CFTR and the
therapeutic composition for treatment are described. The method of
treatment comprises administering an effective amount of genistein,
or genistein analogues and derivatives, to a patient afflicted with
cystic fibrosis.
[0012] In U.S. Patent Publication No. 20040006127, a method for
activation of the chloride is described. Fluorescein and
derivatives have use in the treatment of a disease condition of a
living animal body, including human, which disease is responsive to
the activation of the CFTR chloride channels, for instance cystic
fibrosis, disseminated brocheiectasis, pulmonary infections,
chronic pancreatitis, male infertility and long QT syndrome.
[0013] In U.S. Patent Application No. 20080318984, compounds for
correction of the cellular alteration of a mutant CFTR protein and
uses thereof are described. The disclosure provides for
compositions, pharmaceutical preparations and methods for
correcting cellular processing of a mutant-CFTR protein (e.g.,
.DELTA.F508 CFTR) that are notable for the treatment of cystic
fibrosis. The compositions and pharmaceutical preparations of the
disclosure may comprise one or more aminobenzothiazole-containing
compounds, aminoarylthiazole-containing compounds,
quinazolinylaminopyrimidinone-containing compounds,
bisaminomethylbithiazole-containing compounds, or
phenylaminoquinoline-containing compounds, or an analog or
derivative thereof.
[0014] In a publication WO2009051909, compounds that improve the
cellular alteration of a mutant CFTR protein and uses thereof are
described. The disclosure provides compositions, pharmaceutical
preparations and methods for increasing activity of a mutant-CFTR.
The compositions, pharmaceutical preparations and methods are
notable for the study and treatment of disorders associated with
mutant-CFTR, such as cystic fibrosis. The compositions of the
disclosure may comprise one or more bithiazole-containing
compounds, or an analog or derivative thereof.
[0015] Phenylalanine 508 in CFTR protein occurs on the surface of
NBD1 domain of CFTR. Current structural and biophysical studies
reveal no significant differences between wild-domain protein, and
.DELTA.F508 mutant domain that may affect the folding kinetics and
thermodynamic stability of CFTR protein. Solved crystal structures
of both domains show only slight differences in the reorganization
of the amino acids located near the site, which should be occupied
by F508.
[0016] Each of the forgoing patents and publications are
incorporated herein by reference in their entirety.
SUMMARY
[0017] The object of exemplary embodiments is to provide
compositions, pharmaceutical preparations and methods of correcting
the cellular processing of mutant CFTR protein. F508 deletion has
minimal effect on the structure of NBD1 domain as observed in the
results of X-rays, and cannot explain the dramatic difference in
the behavior of mutant and native forms of CFTR protein in the
cell. For the purposes of an exemplary embodiment, the structural
data of both forms of protein were subjected to computer simulation
designed to determine the dynamic properties of NBD1. In an
exemplary embodiment the molecular dynamics methods have been used.
This method is based on an iterative calculation of the
interactions between the atoms forming the simulated system and
solving equations of motion. These simulations (for both studied
forms of NBD1) results in sets of structures that can be adopted by
the target protein according to the initial physical
assumptions--the so-called trajectories.
[0018] Based on the analysis of molecular dynamics trajectories of
the two domains it is possible to isolate a mutant protein
conformation, which differs significantly from the conformational
states adopted by the wild protein. The conformation possesses the
two major pockets on the surface of the protein located on both
sides of the ATP binding site.
[0019] The structure of protein in this conformation was used to
develop compounds for the correction of .DELTA.F508-CFTR
activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 indicated effects of different compounds on iodide
efflux at 1 .mu.M in .DELTA.F508-CFTR HeLa cells.
[0021] FIG. 2 indicates iodide efflux in untreated WT-CFTR HeLa
cells.
[0022] FIG. 3 indicates iodine efflux in .DELTA.F508-CFTR HeLa
cells with forskolin alone, forskolin plus genistein or forskolin
plus other compounds.
[0023] FIG. 4 indicates the impact of correctors on
.DELTA.F508-CFTR maturation and cell localization.
[0024] FIG. 5 indicates synergistic effects of active compounds on
iodide efflux.
[0025] FIG. 6 indicates current-voltage relationship for
cAMP-dependent chloride currents in HeLa cells.
[0026] FIG. 7 indicates effects of different compounds on iodide
efflux.
[0027] FIG. 8 indicates the effect of 73100 plus 118208 molecules
on nasal potential difference (6VTE) in 6F508/M508 mice.
DETAILED DESCRIPTION
[0028] Exemplary embodiments will be described. Various
modifications, adaptations or variations of the exemplary
embodiments described herein may become apparent to those skilled
in the art as such are disclosed. It will be understood that all
such modifications, adaptations or variations that rely upon the
teachings hereof, and through which these teachings have advanced
the art, are considered to be within the scope and spirit of the
disclosure presented herein.
[0029] The methods and compositions of the exemplary embodiments
may suitably comprise, consist of, or consist essentially of the
components, ingredients, elements, steps and process delineations
described herein. The embodiments illustratively disclosed herein
suitably may be practiced in the absence of any element, process
step, or ingredient which is not specifically disclosed herein.
[0030] Unless otherwise stated, all percentages, parts, and ratios
expressed herein are based upon weight of the total
compositions.
[0031] The headings provided herein serve to illustrate, but not to
limit the teachings herein in any way or manner.
[0032] An exemplary embodiment is a compound of general formula
(I):
##STR00001## [0033] its tautomers, E and Z geometrical isomers,
optically active forms such as enantiomers, diastereomers and their
racemate forms or a mixture of stereoisomeric forms or its
pharmaceutically acceptable salts thereof or complexes thereof;
[0034] wherein Z.sup.1 is independently selected from the group
consisting of: [0035] --C.sub.nH.sub.(2n)--, which is branched or
unbranched wherein n is an integer from 1 to 5;
--C.sub.nH.sub.(2n-2)-- in E or Z geometrical conformation which is
branched or unbranched wherein n is an integer from 2 to 5;
--C.sub.nH.sub.(2n-4)-- which is branched or unbranched wherein n
is an integer from 2 to 5; --CR'H--, --C.sub.2H.sub.3R'--, E or
Z--C.sub.2HR'--, --C.sub.3H.sub.5R'--, E or Z--C.sub.3H.sub.3R'--,
--OCH.sub.2--, --CH.sub.2O--, --NR''CH.sub.2--, --CH.sub.2NR''--;
wherein R' is independently selected from the group consisting of:
--H, halogen, --NH.sub.2, --OH, --CN, CF.sub.3, --CHF.sub.2,
--CH.sub.2F, --SH, --SCN, --CH.sub.3, --C.sub.2H.sub.5; wherein R''
is independently selected from the group consisting of: --H,
--CH.sub.3, --C.sub.2H.sub.5; wherein R.sup.1 and R.sup.2 are
independently selected from the group consisting of aromatic ring
or heteroaromatic ring, as a modulator of a mutant CFTR protein for
use in the treatment of cystic fibrosis. R.sup.1 and R.sup.2 are
independently selected from the group of sub-formula (Ia):
[0035] ##STR00002## [0036] wherein A.sub.1, A.sub.2, A.sub.3,
A.sub.4, A.sub.5, A.sub.6 is independently selected N or C atoms
wherein ring contain 0-3 nitrogen atoms; [0037] wherein E.sup.1,
E.sup.2, E.sup.3, E.sup.4, E.sup.5 represents optional
substituents, which are selected from: --OR.sub.B,
--OC(.dbd.O)R.sub.C, --OC(.dbd.O)OR.sub.B,
--OC(.dbd.O)N(R.sub.A)R.sub.A', --C(.dbd.O)R.sub.C,
--C(.dbd.O)N(R.sub.A)R.sub.A', --C(.dbd.O)N(OR.sub.B)R.sub.A,
--C(.dbd.O)OR.sub.B, --C(.dbd.S)R.sub.C,
--C(.dbd.O)C(.dbd.O)R.sub.C, --CH.sub.2OR.sub.B,
--CH.sub.2CH.sub.2OR.sub.B, --CH.sub.2N(R.sub.A)R.sub.A',
--CH.sub.2CH.sub.2N(R.sub.A)R.sub.A', --CH.sub.2OCH.sub.2R.sub.C,
--CH.sub.2N(R.sub.A)CH.sub.2R.sub.C, --SR.sub.D,
--S(.dbd.O)R.sub.D, --SO.sub.2R.sub.D,
--SO.sub.2N(R.sub.A)R.sub.A', --SO.sub.3R.sub.B,
--N(R.sub.A)C(.dbd.O)R.sub.C, --N(R.sub.A)C(.dbd.O)OR.sub.B,
--N(R.sub.A)C(.dbd.O)N(R.sub.A')R.sub.A'',
--N(R.sub.A)SO.sub.2R.sub.D,
--N(R.sub.A)SO.sub.2N(R.sub.A')R.sub.A'', --N(R.sub.A)R.sub.A',
--N(R.sub.A)C(.dbd.O)R.sub.C, --N(R.sub.A)C(.dbd.O)OR.sub.B,
--N(R.sub.A)N(R.sub.A')R.sub.A'',
--N(R.sub.A')N(R.sub.A)C(.dbd.O)R.sub.C, --NO.sub.2, --CN,
--CF.sub.3, --CHF.sub.2, --CH.sub.2F, --NH.sub.2, --SCN,
--SO.sub.2CN, --F, Cl, --Br, --I, --PO.sub.3H.sub.2,
--OPO.sub.3H.sub.2, --C.sub.nH.sub.2nR.sub.C which is branched or
unbranched wherein n is an integer from 1 to 5;
--C.sub.nH.sub.(2n-2)R.sub.C in E or Z geometrical conformation
which branched or unbranched wherein n is an integer from 2 to 5;
--C.sub.nH.sub.(2n-4)R.sub.C which is branched or unbranched
wherein n is an integer from 2 to 5; [0038] wherein R.sub.A,
R.sub.A', R.sub.A'' are each independently selected from the group
consisting of: --H, lower alkyl group, --CN, --CF.sub.3,
--CHF.sub.2, --CH.sub.2F, --OH; [0039] wherein R.sub.B is
independently selected from the group consisting of: --H, lower
alkyl group, --CN, --CF.sub.3, --CHF.sub.2, --CH.sub.2F,
--CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2I; [0040] wherein R.sub.C is
independently selected from the group consisting of: --H, lower
alkyl group, --CN, --CF.sub.3, --CHF.sub.2, --CH.sub.2F,
--CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2I, --F, --Cl, --Br, --I,
--NH.sub.2, [0041] wherein R.sub.D is independently selected from
the group consisting of: --H, lower alkyl group;
[0042] An exemplary embodiment of the compound is represented by
the following structures:
##STR00003##
[0043] An exemplary embodiment of the compound has effect on mutant
CFTR protein, wherein said CFTR mutation is a mutation
.DELTA.F508-CFTR, or another mutation of class II and where a
mutation .DELTA.F508-CFTR, or another mutation of class II are
involved in CFTR protein malfunction.
[0044] In an exemplary embodiment the CFTR protein malfunction
occurs in the protein associated with the disease cystic
fibrosis.
[0045] A further exemplary embodiment is a modulator according to
the above, for use in the treatment of cystic fibrosis wherein it
has effect on CFTR-dependent ion transport across cellular membrane
and/or it has the ability to increase the number of mutant CFTR
proteins that reach the cell surface.
[0046] An exemplary embodiment is used in the treatment of cystic
fibrosis wherein it has stabilizing effect on the structure of the
mutant CFTR protein and/or blocks the interaction with cellular
proteins responsible for the premature degradation of mutant
CFTR
[0047] An exemplary embodiment is used in the treatment of cystic
fibrosis wherein it has effect on mutant CFTR protein, wherein said
CFTR mutation is a mutation .DELTA.F508-CFTR, or another mutation
of class II.
[0048] An exemplary embodiment are compounds, modulators of a
mutant CFTR protein, of general formula (II):
##STR00004##
its tautomers, E and Z geometrical isomers, optically active forms
such as enantiomers, diastereomers and their racemate forms or a
mixture of stereoisomeric forms or its pharmaceutically acceptable
salts thereof or complexes thereof; wherein Q.sup.1 and Q.sup.2 are
independently selected from the group consisting of: C, CH, N, NH;
wherein A is a fused five-membered ring having 0-3 independently
selected heteroatoms wherein the heteroatoms comprise nitrogen,
sulfur or oxygen; wherein R.sup.4, R.sup.5 and R.sup.6 represent
optional substituents, which are independently selected from:
--OR.sub.B, --OC(.dbd.O)R.sub.C, --OC(.dbd.O)OR.sub.B,
--OC(.dbd.O)N(R.sub.A)R.sub.A', --C(.dbd.O)R.sub.C,
--C(.dbd.O)N(R.sub.A)R.sub.A', --C(.dbd.O)N(OR.sub.B)R.sub.A,
--C(.dbd.O)OR.sub.B, --C(.dbd.S)R.sub.C,
--C(.dbd.O)C(.dbd.O)R.sub.C, --CH.sub.2OR.sub.B,
--CH.sub.2CH.sub.2OR.sub.B, --CH.sub.2N(R.sub.A)R.sub.A',
--CH.sub.2CH.sub.2N(R.sub.A)R.sub.A', --CH.sub.2OCH.sub.2R.sub.C,
--CH.sub.2N(R.sub.A)CH.sub.2R.sub.C, --SR.sub.D,
--S(.dbd.O)R.sub.D, --SO.sub.2R.sub.D,
--SO.sub.2N(R.sub.A)R.sub.A', --SO.sub.3R.sub.B,
--N(R.sub.A)C(.dbd.O)R.sub.C, --N(R.sub.A)C(.dbd.O)OR.sub.B,
--N(R.sub.A)C(.dbd.O)N(R.sub.A')R.sub.A'',
--N(R.sub.A)SO.sub.2R.sub.D,
--N(R.sub.A)SO.sub.2N(R.sub.A')R.sub.A'', --N(R.sub.A)R.sub.A',
--N(R.sub.A)C(.dbd.O)R.sub.C, --N(R.sub.A)C(.dbd.O)OR.sub.B,
--N(R.sub.A)N(R.sub.A')R.sub.A'',
--N(R.sub.A')N(R.sub.A)C(.dbd.O)R.sub.C, --CN, --CF.sub.3,
--CHF.sub.2, --CH.sub.2F, --NH.sub.2, --SCN, --SO.sub.2CN, --F, Cl,
--Br, --I, --PO.sub.3H.sub.2, --OPO.sub.3H.sub.2, which may be
optionally preceded by: --C.sub.nH.sub.(2n-1)R.sub.C which is
branched or unbranched wherein n is an integer from 1 to 4;
--C.sub.nH.sub.(2n-3)R.sub.C in E or Z geometrical conformation
which is branched or unbranched wherein n is an integer from 2 to
5; --C.sub.nH.sub.(2n-5)R.sub.C which is branched or unbranched
wherein n is an integer from 2 to 5; wherein Z.sup.2 is selected
from: a single bond, --N(R.sub.A')--, --S--, --S-alkil-, --O--,
--O-alikil-, --C(.dbd.O)--, --S(.dbd.O)--, --OC(.dbd.O)--,
--C(.dbd.O)N(R.sub.A')--, --OC(.dbd.O)N(R.sub.A')--,
--C(.dbd.O)O--, --SO.sub.2--, --SO.sub.2N(R.sub.A')--,
--N(R.sub.A')SO.sub.2--, --N(R.sub.A')SO.sub.2N(R.sub.A'')--,
--CH.sub.2O--, --N(R.sub.A')C(.dbd.O)--, --N(R.sub.A')C(.dbd.O)O--,
--N(R.sub.A')C(.dbd.O)N(R.sub.A'')--, --C(.dbd.O)C(.dbd.O)--,
--N(R.sub.A')C(.dbd.O)O--, --N(R.sub.A')N(R.sub.A'')--,
--N(R.sub.A')N(R.sub.A'')C(.dbd.O)--,
--C(.dbd.O)N(R.sub.A')N(R.sub.A)--, --CH.sub.2N(R.sub.A')--,
--CH.sub.2CH.sub.2O--, --CH.sub.2CH.sub.2N(R.sub.A')--,
--CH.sub.2OCH.sub.2--, --CH.sub.2N(R.sub.A')CH.sub.2--,
--C.sub.nH.sub.2n which is branched or unbranched wherein n is an
integer from 1 to 5; --C.sub.nH.sub.(2n-2) in E or Z geometrical
conformation which is branched or unbranched wherein n is an
integer from 2 to 5; --C.sub.nH.sub.(2n-4) which is branched or
unbranched wherein n is an integer from 2 to 5; wherein R.sup.7 are
independently selected from the group consisting of: --H, aromatic
ring or heteroaromatic ring; wherein Z.sup.3 is selected from: a
single bond, double bond, --N(R.sub.A')--, --S--, --S-alkyl-,
--O--, --O-alkyl- --C(.dbd.O)--, --C(.dbd.S)--, --OC(.dbd.O)--,
--C(.dbd.O)N(R.sub.A')--, --OC(.dbd.O)N(R.sub.A')--,
--C(.dbd.O)O--, --SO.sub.2--, --SO.sub.2N(R.sub.A')--,
--N(R.sub.A')SO.sub.2--, --N(R.sub.A')SO.sub.2N(R.sub.A')--,
--CH.sub.2O--, --N(R.sub.A')C(.dbd.O)--, --N(R.sub.A')C(.dbd.O)O--,
--N(R.sub.A')C(.dbd.O)N(R.sub.A')--, --C(.dbd.O)C(.dbd.O)--,
--N(R.sub.A')C(.dbd.O)O--, --N(R.sub.A')N(R.sub.A')--,
--N(R.sub.A')N(R.sub.A'')C(.dbd.O)--,
--C(.dbd.O)N(R.sub.A')N(R.sub.A'')--, --CH.sub.2N(R.sub.A')--,
--CH.sub.2CH.sub.2O--, --CH.sub.2CH.sub.2N(R.sub.A')--,
--CH.sub.2OCH.sub.2--, --CH.sub.2N(R.sub.A')CH.sub.2--,
--C.sub.nH.sub.2n-- which is branched or unbranched wherein n is an
integer from 1 to 5; --C.sub.nH.sub.(2n-2) in E or Z geometrical
conformation which is branched or unbranched wherein n is an
integer from 2 to 5; --C.sub.nH.sub.(2n-4) which is branched or
unbranched wherein n is an integer from 2 to 5; wherein R.sup.8 is
selected from: H, O, S, aromatic ring or heteroaromatic ring;
--C.sub.nH.sub.(2n+1) which is branched or unbranched wherein n is
an integer from 1 to 5; --C.sub.nH.sub.(2n-1) in E or Z geometrical
conformation which is branched or unbranched wherein n is an
integer from 2 to 5; --C.sub.nH.sub.(2n-3) which is branched or
unbranched wherein n is an integer from 2 to 5; wherein R.sub.A,
R.sub.A', R.sub.A'' are each independently selected from the group
consisting of: --H, lower alkyl group, --CN, --CF.sub.3,
--CHF.sub.2, --CH.sub.2F, --OH; wherein R.sub.B is independently
selected from the group consisting of: --H, lower alkyl group,
--CN, --CF.sub.3, --CHF.sub.2, --CH.sub.2F, --CH.sub.2Cl,
--CH.sub.2Br, --CH.sub.2I; wherein R.sub.C is independently
selected from the group consisting of: --H, lower alkyl group,
--CN, --CF.sub.3, --CHF.sub.2, --CH.sub.2F, --CH.sub.2Cl,
--CH.sub.2Br, --CH.sub.2I, --F, --Cl, --Br, --I, --NH.sub.2;
wherein R.sub.D is independently selected from the group consisting
of: --H, lower alkyl group; wherein the 5-membered ring A is moiety
selected from the group consisting of:
##STR00005##
and wherein compound
6-[(4-oxo-1H-quinolin-3-yl)carbonylamino]-1H-indole-4-carboxylic
acid ethyl ester is excluded, as a modulator of a mutant CFTR
protein, for use in the manufacture of a medicament for the
treatment of diseases associated with CFTR protein malfunction.
[0049] An exemplary embodiment of the compound described above has
the general formula (IIa) or (IIb):
##STR00006##
wherein Q.sup.1, Q.sup.2, Q.sup.3, Q.sup.4, Q.sup.5 represent
optional substituents which are independently selected from the
group consisting of: --OR.sub.B, --OC(.dbd.O)R.sub.C,
--OC(.dbd.O)OR.sub.B, --OC(.dbd.O)N(R.sub.A)R.sub.A',
--C(.dbd.O)R.sub.C, --C(.dbd.O)N(R.sub.A)R.sub.A',
--C(.dbd.O)N(OR.sub.B)R.sub.A, --C(.dbd.O)OR.sub.B,
--C(.dbd.S)R.sub.C, --C(.dbd.O)C(.dbd.O)R.sub.C,
--CH.sub.2OR.sub.B, --CH.sub.2CH.sub.2OR.sub.B,
--CH.sub.2N(R.sub.A)R.sub.A', --CH.sub.2CH.sub.2N(R.sub.A)R.sub.A',
--CH.sub.2OCH.sub.2R.sub.C, --CH.sub.2N(R.sub.A)CH.sub.2R.sub.C,
--SR.sub.D, --S(.dbd.O)R.sub.D, --SO.sub.2R.sub.D,
--SO.sub.2N(R.sub.A)R.sub.A', --SO.sub.3R.sub.B,
--N(R.sub.A)C(.dbd.O)R.sub.C, --N(R.sub.A)C(.dbd.O)OR.sub.B,
--N(R.sub.A)C(.dbd.O)N(R.sub.A')R.sub.A'',
--N(R.sub.A)SO.sub.2R.sub.D,
--N(R.sub.A)SO.sub.2N(R.sub.A')R.sub.A'', --N(R.sub.A)R.sub.A',
--N(R.sub.A)C(.dbd.O)R.sub.C, --N(R.sub.A)C(.dbd.O)OR.sub.B,
--N(R.sub.A)N(R.sub.A')R.sub.A'',
--N(R.sub.A')N(R.sub.A)C(.dbd.O)R.sub.C, --NO.sub.2, --CN,
--CF.sub.3, --CHF.sub.2, --CH.sub.2F, --NH.sub.2, --SCN,
--SO.sub.2CN, --F, Cl, --Br, --I, --C.sub.nH.sub.2nR.sub.C which is
branched or unbranched wherein n is an integer from 1 to 5;
--C.sub.nH.sub.(2n-2)R.sub.C in E or Z geometrical conformation
which is branched or unbranched wherein n is an integer from 2 to
5; --C.sub.nH.sub.(2n-4)R.sub.C which is branched or unbranched
wherein n is an integer from 2 to 5; --PO.sub.3H.sub.2,
--OPO.sub.3H.sub.2.
[0050] An exemplary embodiment of the compound may be represented
by the following structure:
##STR00007##
[0051] An exemplary embodiment of the compound has effect on mutant
CFTR protein, wherein said CFTR mutation is a mutation
.DELTA.F508-CFTR, or another mutation of class II and where a
mutation .DELTA.F508-CFTR, or another mutation of class II. are
involved in CFTR protein malfunction.
[0052] In an exemplary embodiment the CFTR protein malfunction
occurs in the protein associated with the disease cystic
fibrosis.
[0053] A further exemplary embodiment is a modulator according to
the above, for use in the treatment of cystic fibrosis wherein it
has effect on CFTR-dependent ion transport across cellular membrane
and/or it has the ability to increase the number of mutant CFTR
proteins that reach the cell surface.
[0054] An exemplary embodiment is used in the treatment of cystic
fibrosis wherein it has stabilizing effect on the structure of the
mutant CFTR protein and/or blocks the interaction with cellular
proteins responsible for the premature degradation of mutant
CFTR
[0055] An exemplary embodiment is used in the treatment of cystic
fibrosis wherein it has effect on mutant CFTR protein, wherein said
CFTR mutation is a mutation .DELTA.F508-CFTR, or another mutation
of class II.
[0056] An exemplary embodiment is a compound of general formula
(III):
##STR00008##
its tautomers, E and Z geometrical isomers, optically active forms
such as enantiomers, diastereomers and their racemate forms or a
mixture of stereoisomeric forms or its pharmaceutically acceptable
salts thereof or complexes thereof; wherein Z.sup.1, Z.sup.2,
Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.6, Z.sup.7 represents optional
substituents, which are selected from substituents consisting at
least one atom selected from the group consisting of: C, N, S, O,
H, P, F, Cl, Br, I; wherein R.sup.4 represents optionally
substituted moiety of formula (IIIa):
##STR00009##
wherein R.sup.5 and R.sup.6 are optional substituents which are
independently selected from the group consisting of: OH, NH.sub.2,
COOH, Cl, Br, I, CH.sub.3, C.sub.2H.sub.5; and having a general
formula (IIIb):
##STR00010##
wherein R.sup.7 is an optional substituent which is independently
selected from the group consisting of: --F, --Cl, --Br, --I,
--CH.sub.3, --C.sub.2H.sub.5; wherein R.sup.8 is an optional
substituent which is independently selected from the group
consisting of: --NH.sub.2, --NHAr, --OH, --CH.sub.2Ar,
--C(.dbd.O)Ar, --OAr; wherein Ar is an aromatic group or
heteroaromatic group; wherein Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4,
Z.sup.5, Z.sup.6, Z.sup.7 represent optional substituents which are
independently selected from the group consisting of: --OR.sub.B,
--OC(.dbd.O)R.sub.C, --OC(.dbd.O)OR.sub.B,
--OC(.dbd.O)N(R.sub.A)R.sub.A', --C(.dbd.O)R.sub.C,
--C(.dbd.O)N(R.sub.A)R.sub.A', --C(.dbd.O)N(OR.sub.B)R.sub.A,
--C(.dbd.O)OR.sub.B, --C(.dbd.S)R.sub.C,
--C(.dbd.O)C(.dbd.O)R.sub.C, --CH.sub.2OR.sub.B,
--CH.sub.2CH.sub.2OR.sub.B, --CH.sub.2N(R.sub.A)R.sub.A',
--CH.sub.2CH.sub.2N(R.sub.A)R.sub.A', --CH.sub.2OCH.sub.2R.sub.C,
--CH.sub.2N(R.sub.A)CH.sub.2R.sub.C, --SR.sub.D,
--S(.dbd.O)R.sub.D, --SO.sub.2R.sub.D,
--SO.sub.2N(R.sub.A)R.sub.A', --SO.sub.3R.sub.B,
--N(R.sub.A)C(.dbd.O)R.sub.C, --N(R.sub.A)C(.dbd.O)OR.sub.B,
--N(R.sub.A)C(.dbd.O)N(R.sub.A')R.sub.A'',
--N(R.sub.A)SO.sub.2R.sub.D,
--N(R.sub.A)SO.sub.2N(R.sub.A')R.sub.A'', --N(R.sub.A)R.sub.A',
--N(R.sub.A)C(.dbd.O)R.sub.C, --N(R.sub.A)C(.dbd.O)OR.sub.B,
--N(R.sub.A)N(R.sub.A')R.sub.A'',
--N(R.sub.A')N(R.sub.A)C(.dbd.O)R.sub.C, --NO.sub.2, --CN,
--CF.sub.3, --CHF.sub.2, --CH.sub.2F, --NH.sub.2, --SCN,
--SO.sub.2CN, --F, Cl, --Br, --I, --C.sub.nH.sub.2nR.sub.C which is
branched or unbranched wherein n is an integer from 1 to 5;
--C.sub.nH.sub.(2n-2)R.sub.C in E or Z geometrical conformation
which is branched or unbranched wherein n is an integer from 2 to
5; --C.sub.nH.sub.(2n-4)R.sub.C which is branched or unbranched
wherein n is an integer from 2 to 5, --PO.sub.3H.sub.2,
--OPO.sub.3H.sub.2; wherein R.sub.A, R.sub.A', R.sub.A'' are each
independently selected from the group consisting of: --H, lower
alkyl group, --CN, --CF.sub.3, --CHF.sub.2, --CH.sub.2F, --OH;
wherein R.sub.B is independently selected from the group consisting
of: --H, lower alkyl group, --CN, --CF.sub.3, --CHF.sub.2,
--CH.sub.2F, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2I; wherein
R.sub.C is independently selected from the group consisting of:
--H, lower alkyl group, --CN, --CF.sub.3, --CHF.sub.2, --CH.sub.2F,
--CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2I, --F, --Cl, --Br, --I,
--NH.sub.2; wherein R.sub.D is independently selected from the
group consisting of: --H, lower alkyl group, as a modulator of a
mutant CFTR protein, for use in the manufacture of a medicament for
the treatment of diseases associated with CFTR protein
malfunction.
[0057] An exemplary embodiment of the compound is represented by
the following structure:
##STR00011##
[0058] An exemplary embodiment of the compound has effect on mutant
CFTR protein, wherein said CFTR mutation is a mutation
.DELTA.F508-CFTR, or another mutation of class II and where a
mutation .DELTA.F508-CFTR, or another mutation of class II are
involved in CFTR protein malfunction.
[0059] In an exemplary embodiment the CFTR protein malfunction
occurs in the protein associated with the disease cystic
fibrosis.
[0060] A further exemplary embodiment is a modulator according to
the above, for use in the treatment of cystic fibrosis wherein it
has effect on CFTR-dependent ion transport across cellular membrane
and/or it has the ability to increase the number of mutant CFTR
proteins that reach the cell surface.
[0061] An exemplary embodiment is used in the treatment of cystic
fibrosis wherein it has stabilizing effect on the structure of the
mutant CFTR protein and/or blocks the interaction with cellular
proteins responsible for the premature degradation of mutant CFTR
An exemplary embodiment is used in the treatment of cystic fibrosis
wherein it has effect on mutant CFTR protein, wherein said CFTR
mutation is a mutation .DELTA.F508-CFTR, or another mutation of
class II.
[0062] An exemplary embodiment is a compound of general formula
(IV):
##STR00012##
its esters, ethers, tautomers, E and Z geometrical isomers,
optically active forms such as enantiomers, diastereomers and their
racemate forms or a mixture of stereoisomeric forms or its
pharmaceutically acceptable salts thereof or complexes thereof;
wherein E.sup.1, E.sup.2 represent substituents which are
independently selected from: H, --CH.sub.3, --C.sub.23/4; wherein E
represents optional substituent selected from: --CI, --F, --Br,
--I, --CF.sub.3, --CHF.sub.2, --CH.sub.2F, --CH.sub.2Cl,
--CH.sub.2Br, --CH.sub.2I, optionally substituted lower alkyl
group;
[0063] An exemplary embodiment of the compound is represented by
the following structure:
##STR00013##
[0064] An exemplary embodiment of the compound has effect on mutant
CFTR protein, wherein said CFTR mutation is a mutation
.DELTA.F508-CFTR, or another mutation of class II and where a
mutation .DELTA.F508-CFTR, or another mutation of class II are
involved in CFTR protein malfunction.
[0065] In an exemplary embodiment the CFTR protein malfunction
occurs in the protein associated with the disease cystic
fibrosis.
[0066] A further exemplary embodiment is a modulator according to
the above, for use in the treatment of cystic fibrosis wherein it
has effect on CFTR-dependent ion transport across cellular membrane
and/or it has the ability to increase the number of mutant CFTR
proteins that reach the cell surface.
[0067] An exemplary embodiment is used in the treatment of cystic
fibrosis wherein it has stabilizing effect on the structure of the
mutant CFTR protein and/or blocks the interaction with cellular
proteins responsible for the premature degradation of mutant
CFTR
[0068] An exemplary embodiment is used in the treatment of cystic
fibrosis wherein it has effect on mutant CFTR protein, wherein said
CFTR mutation is a mutation .DELTA.F508-CFTR, or another mutation
of class II.
[0069] FIG. 1
[0070] The effects of different compounds on iodide efflux at 1
.mu.M in .DELTA.F508-CFTR HeLa cells.
[0071] (a) bar graph showing the peak amplitudes of Fsk/Gsk
dependent iodide effluxes in cells treated by the different drugs
as in A. Values are mean of 3 independent experiments. *p<0.05,
**p<0.01.
[0072] (b) chemical structures of active correctors identified in
silico
[0073] (c) examples of iodide efflux curves obtained in HeLa cells
stably transfected with .DELTA.F508-CFTR and treated for 24 hours
with 10 .mu.M with different compounds. CFTR dependent response was
induced by 10 .mu.M Forskolin (Fsk)+30 .mu.M Genistein (Gsk) as
indicated by 10 the horizontal bar above the traces.
[0074] (d) EC50 was determined for active compounds of pocket 2:
407882 and 73100 and one of pocket 1: 130813, for 118208 EC50 could
not be precisely determined since the maximum of iodide efflux was
not reached even at 100 .mu.M (also shown).
[0075] FIG. 2
[0076] To test whether the compounds exhibit potentiator activity
independent of their effect on CFTR trafficking, we examined iodide
efflux in untreated WT-CFTR HeLa cells. Compounds were added along
with forskolin and their effects were compared to that of forskolin
alone or forskolin plus genistein. Unlike genistein, all tested
molecules induced an I.sup.- efflux greater than that of forskolin
alone.
[0077] FIG. 3
[0078] Potentiation was also tested in .DELTA.F508-CFTR HeLa cells
treated for 2 hours with miglustat to rescue .DELTA.F508-CFTR.
I-efflux was stimulated either with forskolin alone, with forskolin
plus genistein or forskolin plus the different compounds. As shown
in the Figure, only genistein was able to increase efflux,
demonstrating the absence of potentiation activity by The
drugs.
[0079] FIG. 4
[0080] Impact of identified correctors on .DELTA.F508-CFTR
maturation and cell localization.
[0081] (a) Effects of different compounds on CFTR processing.
Representative immunoblots of WT-CFTR and .DELTA.F508-CFTR proteins
of the proteins from HeLa cells treated with 1 .mu.M of the
different compounds for 24 hours with Mab 24-1. The positions of
the mature (band C) and immature (band B) forms of CFTR are
indicated.
[0082] (b) Comparison of relative intensity (C/B+C) for WT-CFTR,
.DELTA.F508-CFTR alone and .DELTA.F508-CFTR after correction with
our molecules.
[0083] (c) Effects of the different compounds used at 1 .mu.M on
CFTR localization. Confocal imaging showing the plasma membrane
localisation of WT-CFTR and intracellular localisation of
.DELTA.F508-CFTR. The effect of drugs is illustrated in panels c to
f. Bars: 20 .mu.M. Arrows indicate staining of CFTR at the plasma
membrane.
[0084] FIG. 5
[0085] Synergistic effect of active compounds on iodide efflux
tested at 1 .mu.M.
[0086] (a) Iodide efflux in response to 1 .mu.M Forskolin (Fsk)+30
M Genistein (Gsk) as indicated by the horizontal bar above the
traces, for cells treated for 24 h with a tested compounds alone
and in combination as follows (a) 407882, 118208, (b) 118208, 73100
(c) 407882, 37173. (d) bar graph showing the peak amplitudes of
Fsk/Gst dependent iodide effluxes in cells treated by the different
drugs as in. Values are mean of 3 independent experiments.
*p<0.05, **p<0.01
[0087] FIG. 6
[0088] (a)/(b) Current-voltage relationship for cAMP-dependent
chloride currents in HeLa cells treated with 407882(12) plus
118208(6) compounds at 1 .mu.M.
[0089] FIG. 7
[0090] The effects of different compounds on iodide efflux at 1
.mu.M in an epithelial serous cell line derived from a .DELTA.F508
CF patient (CF-4KM) cells. The concentration-dependence has been
shown for the most potent molecule 407882
[0091] FIG. 8
[0092] The effect of 73100 plus 118208 molecules on nasal potential
difference (.DELTA.V.sub.TE) in .DELTA.F508/.DELTA.F508 mice. Basal
V.sub.TE values and .DELTA.V.sub.TE changes induced by perfusion of
nasal epithelium with 100 .mu.M amiloride, .DELTA.V.sub.TEamil were
similar in mice treated with the two molecules or with liposomes
alone. Perfusion of low Cl.sup.- solution in 3 out of 5 mice
hyperpolarized V.sub.TE by more than 2 mV
(.DELTA.V.sub.TEamil-lowCl) i.e. the threshold value established by
us as significant effect of treatment. The CFTR-related current
unmasked by CFTR inhibitor I.sub.Inh172 represents about 30% of
(.DELTA.V.sub.TEamil-lowCl) (data not shown).
[0093] For a better understanding of some exemplary embodiments,
the examples of the subject matter are disclosed below.
EXAMPLES
Materials and Methods
Antibodies
[0094] The following antibodies were used: MAB25031 (clone 24-1,
R&D systems, USA) and MM13-4 (Upstate,) monoclonal antibodies
(mAb) for CFTR detection; Fluorescent secondary antibodies Alexa
594 and 488 were purchased from Molecular Probes (Cergy Pontoise,
France)
Cell Culture
[0095] Stably transfected HeLa cells (with pTracer plasmid alone as
a control (pTracer) or expressing WT-CFTR (spTCF-WT),
.DELTA.F508-CFTR s(pTCF-F508del) were provided by Pascale Fanen
(Inserm U.468, Creteil, France) and grown as described in Bobadilla
J L, Macek M, Jr., Fine J P, Farrell P M. Cystic fibrosis: a
worldwide analysis of CFTR mutations--correlation with incidence
data and application to screening. Hum Mutat, 2002 June;
19(6):575-606. Briefly, HeLa cells were grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10%
heat-inactivated FCS, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin and 250 .mu.g/ml zeocin. Cultures were done at
37.degree. C. in a humidified incubator with 5% CO.sub.2. The
expression of WT-CFTR and .DELTA.F508-CFTR in these cells was
verified by immunoprecipitation and immunocytochemistry throughout
the study. Treatments with different molecules (at 1 and 10 .mu.M)
and vehicle were done when cells reached 75% confluence.
[0096] CF-KM4 cell line, obtained by transformation of primary
cultures of CF tracheal gland serous cells homozygous for the
.DELTA.F508 mutation by using the wild-type SV40 virus, were grown
as described elsewhere in the art (Antigny, F. et al. Calcium
homeostasis is abnormal in cystic fibrosis airway epithelial cells
but is normalized after rescue of F508del-CFTR. Cell calcium 43,
175-83(2008)).
Immunoblot Experiments
[0097] Cells cultured in 75 cm.sup.2 flasks were washed twice with
ice cold PBS, scraped in 2 ml PBS and centrifuged at 600 g for 5
min. The pellets were suspended in 300 .mu.l RIPA buffer (50 mM
Tris-HCl, 150 mM NaCl, 1% TritonX-100, 1% Na deoxycholate, 0.1%
SDS, pH 7.5) at 4.degree. C. for 30 min with agitation After
centrifugation at 15000 g for 30 min the supernatants were
processed for immunoblot experiments as previously described in the
art (Baudouin-Legros, M. et al. Control of basal CFTR gene
expression by bicarbonate-sensitive adenylyl cyclase in human
pulmonary cells. Cellular physiology and biochemistry:
international journal of experimental cellular physiology,
biochemistry, and pharmacology 21, 75-86(2008)) with slight
modifications.
[0098] The samples were resolved by 8% SDS-PAGE, transferred onto
PVDF membranes and analysis was performed following manufacturer's
recommendations for the Odyssey infrared imaging system (LI-COR
Biosciences, NE, USA). Blot membranes were blocked with Odyssey
buffer (ScienceTec, Paris, France) for 1 hour and hybridized using
monoclonal anti CFTR Mab24-1 ( 1/1000). The proteins were
visualized by incubation with secondary antibodies ( 1/10000) and
detected using ECL technique as described in, Bensalem, N. et al.
Down-regulation of the anti-inflammatory protein annexin Al in
cystic fibrosis knock-out mice and patients. Molecular &
cellular proteomics: MCP 4, 1591-601(2005).
Immunofluorescence Staining
[0099] HeLa cells grown on glass coverslips were treated as above
and as descrbed in Lipecka, J. et al., Distribution of ClC-2
chloride channel in rat and human epithelial tissues. American
journal of physiology. Cell physiology 282, C805-16(2002). Cells
were fixed with 4% formaldehyde and permeabilized with 0.1% Triton
in PBS. Cells were blocked with 1% bovine serum albumine in
PBS/Triton and incubated at 4.degree. C. overnight with the primary
antibodies, 24-1 (1:300). After washing and blocking with 5% normal
goat serum, cells were incubated with the secondary antibodies.
Glass coverslips were mounted using the Vectashield mounting medium
(Vector laboratories) and examined by confocal laser microscopy
(Zeiss, LSM 510).
Iodide Efflux Experiments
[0100] CFTR chloride channel activity was assayed by measuring
iodide (.sup.125I) efflux from transfected CHO cells as described
previously in the art, Marivingt-Mounir, C, et al., Synthesis, SAR,
crystal structure, and biological evaluation of benzoquinoliziniums
as activators of wild-type and mutant cystic fibrosis transmembrane
conductance regulator channels. J Med Chem, 2004. 47(4): p. 962-72.
Marivingt-Mounir, C, et al., Synthesis, SAR, crystal structure, and
biological evaluation of benzoquinoliziniums as activators of
wild-type and mutant cystic fibrosis transmembrane conductance
regulator channels. J Med Chem, 2004. 47(4): p. 962-72. Cells grown
for 4 days in 96-well plates were washed twice with 2 ml of
modified Earle's salt solution containing 137 mM NaCl, 5.36 mM KCl,
0.4 mM Na.sub.2HPO.sub.4, 0.8 mM MgCl.sub.2, 1.8 mM CaCl.sub.2, 5.5
mM glucose, and 10 mM HEPES, pH 7.4. Cells were then incubated in
the same medium containing 1 mM KI (1 mCi of Na.sup.125I/ml, NEN
Life Science Products) for 30 min at 37.degree. C. After washing,
cells were incubated with 1 ml of modified Earle's salt solution.
After 1 min, the medium was removed to be counted and was quickly
replaced by 1 ml of the same medium. This procedure was repeated
every 1 min for 8 min. The first three aliquots were used to
establish a stable baseline in efflux buffer alone. Medium
containing cocktail aiming to increase intracellular cAMP (10 .mu.M
forskolin and 30 .mu.M genistein) was used for next aliquots in
order to activate CFTR chloride channels. At the end of the
incubation, the medium was recovered, and cells were solubilized in
1 N NaOH. The radioactivity was determined using a g-counter (LKB).
The total amount of .sup.125I (in cpm) at time 0 was calculated as
the sum of cpm counted in each 1-min sample plus the cpm in the
NaOH fraction. The fraction of initial intracellular .sup.125I lost
during each time point was determined, and time-dependent rates of
.sup.125I efflux were calculated according to the art in, Becq, F.,
et al., Development of substituted Benzo[c]quinolizinium compounds
as novel activators of the cystic fibrosis chloride channel. J Biol
Chem, 1999. 274(39): p. 27415-25, from
ln(.sup.125I.sub.t1/.sup.125I.sub.t2)/(t.sub.1-t.sub.2), where
[0101] .sup.125It is the intracellular .sup.125I at time t; and
[0102] t.sub.1 and t.sub.2 are successive time points.
[0103] Curves were constructed by plotting rate of .sup.125I efflux
versus time. Data are presented as the mean.+-.S.E. of n separate
experiments.
[0104] Differences were considered statistically significant using
the Student's t test when the p value was less than 0.05.
Whole Cell Patch-Clamp Recordings
[0105] Technique for patch-clamp recordings in the whole cell
configuration has been described, such as in Hinzpeter, A. et al.
Association between Hsp90 and the ClC-2 chloride channel
upregulates channel function. American journal of physiology. Cell
physiology 290, C45-56(2006) and Tanguy, G. et al. CSN5 binds to
misfolded CFTR and promotes its degradation. Biochimica et
biophysica acta 1783, 1189-99(2008). Stably transfected cells were
plated in 35 mm cell culture plastic Petri dishes that were mounted
on the stage of an inverted microscope. Patch-clamp experiments
were performed at room temperature with an Axopatch 200 A amplifier
controlled by a computer via a digitdata 1440 interface (Axon
Intruments, USA). Pipettes were pulled from hard glass (Kimax 51)
using a Setter micropipette puller and their tips were
fire-polished. Current recordings were performed using the
nystatin-perforated patch clamp configuration. Nystatin stock
solution (50 mg/ml) was prepared daily in DMSO. The stock solution
was diluted (1:250) in the internal solution which was then
sonicated during 1 minute. The internal solution contained the
following (in mM):, 131 NaCl, 2 MgCl.sub.2, and 10 Hepes-Na.sup.+,
pH 7.3, adjusted with NaOH. The bath solution contained (in mM):
150 NaCl, 1 CaCl.sub.2, 1 MgCl.sub.2, 35 sucrose and 10
Hepes-Na.sup.+, pH 7.3, adjusted with NaOH.
[0106] Currents were recorded by application of regular voltage
pulses of 60 mV amplitude during 1 second, from a holding potential
of 0 mV, with an interval of 3 seconds.
[0107] To establish I-V curves, regular voltage pulses were
interrupted by series of 9 voltage jumps (1-s duration each),
toward membrane potentials between -100 and +80 mV. CFTR Cl--
currents were activated with 200 .mu.m 8-(4-chlorophenylthio)-cAMP
sodium salt (CPT-cAMP) plus 100 .mu.M 3-isobutyl-1-methylxanthine
(IBMX).
[0108] When maximal stimulation was reached, cells were bathed with
5 .mu.M of the specific CFTRinhibitor, CFTR.sub.inh-172, added to
the CPT-cAMP solution. CFTR-currents were defined as the
differences in current amplitudes recorded during maximum
stimulation by CPT-cAMP and after inhibition by
CFTR.sub.inh-172.
Nasal Potential Difference (NPD) Measurements
[0109] The method for nasal potential measurement was adapted and
miniaturised from the technique developed for young children as
shown in Sermet-Gaudelus, I. et al. Measurement of nasal potential
difference in young children with an equivocal sweat test following
newborn screening for cystic fibrosis. Thorax 65, 539-44(2010).
Mice were anesthetized by an intraperitoneal injection of ketamine
(133 mg/kg; IMALGENE 1000, MERIAL, France) and xylazine (13.3
mg/kg; Rompun 2%, BayerPharma, France). Mice were positioned on a
45.degree. tilt board and a paper pad was placed under the nose to
avoid mice quelling. A subcutaneous needle was connected to an
Ag.sup.+/AgCl reference electrode by an agar bridge. A double-lumen
polyethylene catheter (0.5 mm diameter) was inserted into one
nostril 4 mm depth. One lumen perfused by a Ringer solution (in mM:
140 NaCl, 6 KCl, 10 Hepes, 10 Glucose, 1 MgCl2, 2 CaCl2, pH
adjusted to 7.4 with NaOH) at 0.15 mL/h is connected to a measuring
Ag.sup.+/AgCl electrode. The two Ag.sup.+/AgCl electrodes were
connected to a high-impedance voltmeter (LOGAN research Ltd, United
Kingdom). The second lumen perfused solution with the following
sequence: (1) Ringer solution, (2) Ringer solution containing
amiloride (inhibitor of Na.sup.+ conductance, 100 .mu.M), (3) Low
Chloride Ringer solution, to unmask Cl.sup.- conductances (in mM:
140 Na gluconate, 6 K gluconate, 10 Hepes, 10 Glucose, 1
MgCl.sub.2, 6 Ca-gluconate, pH adjusted to 7.4 with NaOH), (4) Low
Chloride Ringer solution containing CFTR inhibitor-172 (5 .mu.M,
Calbiochem, Germany) to evaluate the participation of CFTR. Each
solution was perfused at least 3 minutes, and 30 seconds stability
was required before perfusion switch. Steady state transepithelial
potential, V.sub.TE, .DELTA.V.sub.TEAmil (difference between
V.sub.TE and transepithelial potential recorded after perfusion of
amiloride-containing solution), .DELTA.V.sub.TEamilLowCl
(difference between V.sub.TE and transepithelial potential recorded
after perfusion with Low Cl.sup.- plus amiloride-containing
solution) and .DELTA.V.sub.TEamilLowClInh-172 (difference between
V.sub.TE and after addition of CFTR inhibitor to the previous
solution) were the means of 30 values recorded during
stability.
MTT Cell Viability Assay
[0110] To determine cell viability the typical MTT assay was used.
HeLa cells were cultured in a 96-well plate and exposed to varying
concentrations of compounds disclosed herein for 24 h. After
washing, MTT solution and medium were then introduced. After
incubation, the resultant formazan crystals were dissolved in
dimethyl sulfoxide and the absorbance intensity measured by a
microplate reader at 570 nm.
[0111] Yellow MTT is reduced to purple formazan in living cells.
The absorbance of this colored solution can be quantified by
measuring at a certain wavelength by a spectrophotometer. This
conversion can be directly related to the number of viable (living)
cells.
Virtual Screening--Identification of Modulator Compounds
[0112] A database of a low molecular weight compounds was used in
the virtual screening process as a source of hits. Molecular
docking program Dock 6.1 was used to test a conformational space of
small molecules inside two potential binding sites on the protein
surface. Subsequently, all selected ligands and whole complexes
were fully minimized in force field. At each step, a set of scoring
functions was used for rating of potential complexes and
appropriate molecules were selected for experimental tests.
TABLE-US-00001 ##STR00014## 118208/NOP1.6/Pok 1c
2-[(3-nitrophenyl)methylsulfanyl]-3,7- dihydropurin-6-one
##STR00015## 407882/NOP2.6/Pok 2f
2-[hydroxy(phenyl)phosphoryl]ethyl- phenylphosphinic acid
##STR00016## 130813/NOP1.2/Pok 1b 4-((6-chloro-2-methoxy-9-
acridinyl)amino)-2-((4-methyl-1- piperazinyl)methyl)phenol
##STR00017## 73100/Pok2e 2-(9H-flouren-2- ylcarbamoyl)terephthalic
acid
Results
Effect of Drugs on Iodide, I--, Efflux
[0113] To test drug correction of .DELTA.F508-CFTR trafficking and
function we evaluated halide permeability by a macroscopic assay
using a robotic cell-based methodology using the I efflux
technique. In the first series of experiments, the potential
corrector effects were tested by 24 hour pre-treatment of
.DELTA.F508-CFTR HeLa cells with all compounds at 1 M followed by
measurements of cAMP-dependent radiolabel iodide efflux. Treatments
with compounds 130813 and 118208 on pocket 1 and 73100 and 407882
on pocket 2, lead to significant increase of cAMP-stimulated
radiolabel iodide efflux (FIG. 1a), the most potent being 407882.
At this low dose (1 .mu.M) the increase in the cAMP-stimulated
efflux was lower than that observed using 100 .mu.M of the known
corrector miglustat (27). Examples of I.sup.- efflux stimulation
after treatment with each of the four active compounds are
illustrated in FIG. 1b. cAMP-stimulated F efflux was completely
prevented when experiments were performed in the presence of the
CFTR channel blocker CFTR.sub.inh-172.
[0114] We further tested the effect of the four compounds in a wide
range of concentrations and determined EC.sub.50 for pocket 1
compound 130813, and two pocket 2 compounds 407882 and 73100 at 1
.mu.M, 10 .mu.M and 844 nM, respectively (FIG. 1c). The EC.sub.50
for pocket 1-118208 could not be precisely determined since the
maximum iodide efflux was not reached even at 100 .mu.M (FIG. 1d).
Notably, the effect of compound 407882 could be increased by 3-fold
when used at 10 .mu.M, reaching a stimulated efflux comparable to
the value observed for WT-CFTR (FIG. 2).
[0115] To test whether the compounds exhibit potentiator activity
independent of their effect on CFTR trafficking, we examined iodide
efflux in untreated WT-CFTR HeLa cells. Compounds were added along
with forskolin and their effects were compared to that of forskolin
alone or forskolin plus genistein. Unlike genistein, all tested
molecules induced an I.sup.- efflux greater than that of forskolin
alone (FIG. 2). Potentiation was also tested in .DELTA.F508-CFTR
HeLa cells treated for 2 hours with miglustat to rescue
.DELTA.F508-CFTR. I.sup.- efflux was stimulated either with
forskolin alone, with forskolin plus genistein or forskolin plus
the different compounds. As shown in FIG. 3, only genistein was
able to increase efflux, demonstrating the absence of potentiation
activity by our drugs.
Effect of Drugs on CFTR Maturation
[0116] The efficacy of the four compounds as correctors for
.DELTA.F508-CFTR trafficking was further validated by
immunoblotting. We assumed that detection of a fully glycosylated
band C suggests correct processing of .DELTA.F508-CFTR. A
representative immunoblot is shown in FIG. 4a. Anti-CFTR antibodies
detect two bands in proteins derived from WT-CFTR cells, (line
WT-CFTR in FIG. 4a). The diffuse band of approximately 170 kDa
(band C) corresponds to a mature, fully glycosylated protein that
has processed through the Golgi apparatus. The band below of about
145 kDa corresponds to the immature core-glycosylated protein
located in the endoplasmic reticulum. In .DELTA.F508-CFTR
expressing cells, only the immature protein is detectable (line
.DELTA.F508 in FIG. 4a). Band C was clearly detectable in cells
treated with 1 .mu.M of compound 407882 as compared to untreated
cells, whereas the signal at 170 kDa was not different from DMSO
treatment in cells treated with 1 .mu.M of compounds 118208, or
130813 or very slightly increased in cells treated by 1 .mu.M of
compound 73100. None of the compounds modified total protein
expression. The relative abundance of mature CFTR, expressed as the
ratio of band C to band C+ band B is shown in FIG. 4b. Only
compound 407882 increased significantly the relative abundance of
mature CFTR.
Effect of Drugs on CFTR Immunolocalization
[0117] FIG. 4c shows typical CFTR staining at the plasma membrane
in WT-CFTR expressing HeLa cells whereas .DELTA.F508-CFTR was found
throughout the cytoplasm. Treatment of cells for 24 hours with 1
.mu.M of 407882 resulted in a clear CFTR staining at or near the
plasma membrane, indicating rescue of .DELTA.F508-CFTR trafficking
in agreement with immunoblot experiments. When cells were treated
by each of the three other compounds, 118208, 73100 or 130813, a
discrete punctuate staining at the plasma membrane was observed in
a small fraction of cells, as illustrated for compound 118208 in
FIG. 4c.
Combined Effect of Compounds Binding to Different Pockets.
[0118] If two compounds are able to correct .DELTA.F508-CFTR by
binding to the same protein conformation but at different surface
cavities their effects could be additive or synergistic. We tested
this hypothesis by two independent types of assays, namely iodide
efflux and patch clamp. The results from iodide permeability tests
(FIG. 5) showed that combined treatment of cells with compounds
118208 plus 407882 (FIG. 5a) or with 118208 plus 73100 (FIG. 5b) at
1 .mu.M of each, leads to greater cAMP-dependent anion fluxes than
those observed with any of the molecules alone. In this series of
experiments the compound 37173 (FIG. 5c) was used as a control as
it did not induce any cAMP-stimulated iodide efflux at the same
concentration. As shown in FIG. 5c, co-treatment of
.DELTA.F508-CFTR HeLa cells with 37173 plus 407882 induced
cAMP-stimulated iodide efflux with an amplitude similar to 407882
treatment alone. By contrast, co-treatment with compounds 118208
and 407882 induced iodide efflux with an amplitude equal to the sum
of effluxes induced by each compound, whereas a slight synergistic
effect was observed after treatment by 118208 plus 73100.
[0119] The activity of the different compounds was also evaluated
in patch-clamp experiments. FIG. 6a summarizes the mean values of
current amplitudes recorded at -60 mV in the different experimental
conditions. CFTR-related current density (I.sub..DELTA.F508-CFTR;
pA/pF) is defined as cAMP-stimulated current minus the current
recorded after inhibition by CFTR inh-172 at 5 .mu.M, and
normalized to cell capacitance. I.sub..DELTA.F508-CFTR was very low
in untreated cells and stimulated by around 3-fold when cells were
cultured at 27.degree. C. for 24 hours before recordings. Treatment
of cells for 24 h with 1 .mu.M of either 118208, 407882 or 73100
alone, did not increase current amplitude as compared to their
respective controls (data not shown). However, 24 h pre-treatment
with 1 .mu.M of 118208 plus 407882 or 118208 plus 73100 showed a
significant increase in I.sub..DELTA.F508-CFTR. Examples of linear
I/V plots from cells pretreated by 118208 plus 407882 before
stimulation, in the presence of cptcAMP+IBMX and after inhibition
by CFTRinh-172 are shown in FIG. 6b.
Effects of 407882 and 118208 on CF-4KM Cells
[0120] The effects of the four molecules active in HeLa cells were
next tested on CFTR-dependent iodide efflux in an epithelial serous
cell line derived from a .DELTA.F508 CF patient (CF-KM4) expressing
low amounts of endogenous .DELTA.F508-CFTR. In these epithelial
cells compounds 407882 and 118208 were still able to induce
significant cAMP-dependent iodide efflux (FIG. 7). However, it must
be noted that 2 molecules correcting .DELTA.F508-CFTR in Hela cells
(130813 and 73100) were not active in this cell line.
Effects of 73100 Plus 118208 on Nasal Potential Difference in
.DELTA.F508 Mice.
[0121] Our results in cells suggested that the pairs of molecules
acting on different pockets display additive correcting effects. To
test if these molecules are active in vivo, nasal potential
difference (.DELTA.V.sub.TE) was monitored (as in Sermet-Gaudelus,
I. et al., Measurement of nasal potential difference in young
children with an equivocal sweat test following newborn screening
for cystic fibrosis. Thorax 65, 539-44(2010)) in
.DELTA.F508/.DELTA.F508 mice treated intranasally for 24 hours with
30 l of 73100 plus 118208 molecules (0.1 .mu.mol each) embedded in
liposomes (5:1) or with liposomes alone. In .DELTA.F508 mice, basal
V.sub.TE values and .DELTA.V.sub.TE changes induced by perfusion of
nasal epithelium with 100 .mu.M amiloride, .DELTA.V.sub.TEamil were
similar in mice treated with the two molecules or with liposomes
alone. By contrast, perfusion of low Cl.sup.- solution in 3 out of
5 mice hyperpolarized V.sub.TE by more than 2 mV
(.DELTA.V.sub.TEamil-lowCl) i.e. the threshold value established by
us as significant effect of treatment (manuscript in preparation).
The CFTR-related current unmasked by CFTR inhibitor I.sub.Inh172
represents about 30% of (.DELTA.V.sub.TEamil-lowCl) (data not
shown).
[0122] The following references are incorporated herein in their
entirety:
[0123] 1. Ollero M, Brouillard F, Edelman A. Cystic fibrosis enters
the proteomics scene: new answers to old questions. Proteomics,
2006 July; 6(14):4084-99. [0124] 2. Riordan J R, Rommens J M, Kerem
B, Alon N, Rozmahel R, Grzelczak Z, et al. Identification of the
cystic fibrosis gene: cloning and characterization of complementary
DNA. Science, 1989 Sep. 8; 245(4922):1066-73. [0125] 3. Rommens J
M, Iannuzzi M C, Kerem B, Drumm M L, Melmer G, Dean M, et al.
Identification of the cystic fibrosis gene: chromosome walking and
jumping. Science, 1989 Sep. 8; 245(4922): 1059-65. [0126] 4.
Castellani C, Cuppens H, Macek M, Jr., Cassiman J J, Kerem E, Durie
P, et al. Consensus on the use and interpretation of cystic
fibrosis mutation analysis in clinical practice. J Cyst Fibros,
2008 May; 7(3): 179-96. [0127] 5. Cystic Fibrosis Mutation Database
http://www.genet.sickkids.on.ca/cftr/app. [0128] 6. Welsh M J,
Smith A E. Molecular mechanisms of CFTR chloride channel
dysfunction in cystic fibrosis. Celi, 1993 Jul. 2; 73(7): 1251-4.
[0129] 7. Wilschanski M, Zielenski J, Markiewicz D, Tsui L C, Corey
M, Levison H, et al. Correlation of sweat chloride concentration
with classes of the cystic fibrosis transmembrane conductance
regulator gene mutations. J Pediatr, 1995 November; 127(5):705-10.
[0130] 8. Ward C L, Omura S, Kopito R R. Degradation of CFTR by the
ubiquitin-proteasome pathway. Cell, 1995 Oct. 6; 83(1):121-7.
[0131] 9. Bobadilla J L, Macek M, Jr., Fine J P, Farrell P M.
Cystic fibrosis: a worldwide analysis of CFTR mutations-correlation
with incidence data and application to screening. Hum Mutat, 2002
June; 19(6):575-606. [0132] 10. Lewis H A, Zhao X, Wang C, Sauder J
M, Rooney I, Noland B W, et al. Impact of the delt.DELTA.F508
mutation in first nucleotide-binding domain of human cystic
fibrosis transmembrane conductance regulator on domain folding and
structure. J Biol Chem, 2005 Jan. 14; 280(2): 1346-53. [0133] 11.
Schwiebert E M, Benos D J, Egan M E, Stutts M J, Guggino W B. CFTR
is a conductance regulator as well as a chloride channel. Physiol
Rev, 1999 January; 79(1 Suppl):S145-66. [0134] 12. Reddy M M, Light
M J, Cjuinton P M. Activation of the epithelial Na+ channel (ENaC)
requires CFTR Cl-channel function. Nature, 1999 Nov. 18;
402(6759):301-4. [0135] 13. Ahmed N, Corey M, Forstner G, Zielenski
J, Tsui L C, Ellis L, et al. Molecular consequences of cystic
fibrosis transmembrane regulator (CFTR) gene mutations in the
exocrine pancreas. Gut, 2003 August; 52(8): 1159-64. [0136] 14.
Riordan J R. Assembly of functional CFTR chloride channels. Annu
Rev Physiol, 2005; 67:701-18. [0137] 15. Allen M P, Tildesley D J.
Computer simulation of liquids: Oxford, Clarendon Press; 1987.
[0138] 16. Jungas, T., et al., Glutathione levels and BAX
activation during apoptosis due to oxidative stress in cells
expressing wild-type and mutant cystic fibrosis transmembrane
conductance regulator. J Biol Chem, 2002. 277(31): p. 27912-8.
[0139] 17. Antigny, F. et al. Calcium homeostasis is abnormal in
cystic fibrosis airway epithelial cells but is normalized after
rescue of F508del-CFTR. Cell calcium 43, 175-83(2008). [0140] 18.
Baudouin-Legros, M. et al. Control of basal CFTR gene expression by
bicarbonate-sensitive adenylyl cyclase in human pulmonary cells.
Cellular physiology and biochemistry: international journal of
experimental cellular physiology, biochemistry, and pharmacology
21, 75-86(2008). [0141] 19. Bensalem, N. et al. Down-regulation of
the anti-inflammatory protein annexin Al in cystic fibrosis
knock-out mice and patients. Molecular & cellular proteomics:
MCP 4, 1591-601(2005). [0142] 20. Lipecka, J. et al. Distribution
of ClC-2 chloride channel in rat and human epithelial tissues.
American journal of physiology. Cell physiology 282, C805-16(2002).
[0143] 21. Marivingt-Mounir, C, et al., Synthesis, SAR, crystal
structure, and biological evaluation of benzoquinoliziniums as
activators of wild-type and mutant cystic fibrosis transmembrane
conductance regulator channels. J Med Chem, 2004. 47(4): p. 962-72.
[0144] 22. Becq, F., et al., Development of substituted
Benzo[c]quinolizinium compounds as novel activators of the cystic
fibrosis chloride channel. J Biol Chem, 1999. 274(39): p. 27415-25.
[0145] 23. Hinzpeter, A. et al. Association between Hsp90 and the
ClC-2 chloride channel upregulates channel function. American
journal of physiology. Cell physiology 290, C45-56(2006). [0146]
24. Tanguy, G. et al. CSN5 binds to misfolded CFTR and promotes its
degradation. Biochimica et biophysica acta 1783, 1189-99(2008).
[0147] 25. Sermet-Gaudelus, I. et al. Measurement of nasal
potential difference in young children with an equivocal sweat test
following newborn screening for cystic fibrosis. Thorax 65,
539-44(2010). [0148] 26. Norez, C. et al. Maintaining low Ca2+
level in the endoplasmic reticulum restores abnormal endogenous
F508del-CFTR trafficking in airway epithelial cells. Traffic
(Copenhagen, Denmark) 7, 562-73(2006). [0149] 27. Norez, C. et al.
Rescue of functional delF508-CFTR channels in cystic fibrosis
epithelial cells by the alpha-glucosidase inhibitor miglustat. FEBS
letters 580, 2081-6(2006).
[0150] Of course these methods are exemplary and alterations
thereto are possible by those having skill in the relevant
technology.
[0151] Thus the example embodiments and arrangements achieve
improved capabilities, eliminate difficulties encountered in the
use of prior methods and systems, and attain the desirable results
described herein.
[0152] In the foregoing description, certain terms have been used
for brevity, clarity and understanding. However, no unnecessary
limitations are to be implied therefrom because such terms are used
for descriptive purposes and are intended to be broadly
construed.
[0153] Moreover the descriptions and illustrations herein are by
way of examples and the inventive scope is not limited to the
features shown and described.
[0154] Further, it should be understood that features and/or
relationships associated with one embodiment can be combined with
features and/or relationships from other embodiments. That is,
various features and/or relationships from various embodiments can
be combined in further embodiments. The inventive scope of the
disclosure is not limited to only the embodiments shown or
described herein.
[0155] Having described the features, discoveries and principles of
the exemplary embodiments, the manner in which they are utilized
and carried out, and the advantages and useful results attained,
the new and useful arrangements, combinations, methodologies,
structures, devices, elements, combinations, operations, processes
and relationships are set forth in the appended claims.
* * * * *
References