Patent Application: US-201214125109-A

Abstract:
the invention relates to the use of 4 , 6 , 4 ′- trimethylangelicin and structural analogues thereof to prepare a medicament for the treatment of cystic fibrosis with the primary objective of correcting the defective cftr in a sub - group of cystic fibrosis patients consisting of patients carrying the f508de1 - cftr mutation .

Description:
we have discovered that 4 , 6 , 4 ′- trimethylangelicin ( tma ) is a powerful “ corrector ” of f508del - cftr . this effect is unexpected , and has not been previously described for linear and angular psoralens , which have been studied for decades , including on cystic fibrosis cells . the same molecule ( tma ), therefore , possesses three activities , all of which are of interest for the treatment of cystic fibrosis : anti - inflammatory activity , f508del - cftr “ potentiator ” activity and f508del - cftr “ corrector ” activity . the structural formula of tma , compared with angelicin and with the compound 8 - mop and 5 - mop ( selected by way of example from the linear psoralens ), is as follows : earlier results demonstrated that tma inhibits the accumulation of il - 8 mrna in bronchial epithelial cells ib3 - 1 ( 12 ) after infection with p . aeruginosa ( 10 ) and , in parallel , that it possesses an effect as “ potentiator ” on f508del - cftr . the activity of 4 , 6 , 4 ′- tma as “ corrector ” of f508del - cftr , which underlies the invention , is unexpected in view of the known therapeutic uses of structural analogues of 4 , 6 , 4 ′- tma ( 13 - 24 ). the chemical synthesis of 4 , 6 , 4 ′- tma , and of the psoralens in general , has been described by various research groups ( see , for example , references 15 - 18 ). a first subject of the present invention is therefore 4 , 6 , 4 ′- trimethylangelicin for use as a “ corrector ” of f508del - cftr either in heterozygous or homozygous state in patients suffering from cystic fibrosis . moreover , a combined treatment with various modulators of the mutated protein f508del - cftr could increase the biological and clinical response to the treatment . a second subject of the present invention is therefore a combination of 4 , 6 , 4 ′- trimethylangelicin ( tma ) with at least one further molecule with a modulating action such as another “ corrector ” or “ potentiator ” of mutated cftr , such as the corrector 3 -{ 6 -{[ 1 -( 2 , 2 - difluoro - 1 , 3 - benzodioxol - 5 - yl ) cyclopropanecarbonyl ] amino }- 3 - methylpyridin - 2 - yl } benzoic acid ( vx - 809 ) and the “ potentiator ” n -( 2 , 4 - di - tert - butyl - 5 - hydroxyphenyl ) 4 - oxo - 1 , 4 - dihydroquinoline - 3 - carboxamide ( vx - 770 ). tma can be administered by suitable delivery systems , to facilitate the correcting / enhancing effect on f508del - cftr compared with the anti - inflammatory effect , depending on the patient &# 39 ; s genotype / phenotype . tma , either alone or combined with said medicaments , can be administered orally or by inhaler ; examples of formulations suitable for oral administration include capsules , tablets , syrups , solutions or drinkable suspensions and similar conventional dosage forms . dosage forms suitable for administration by inhaler include solutions , suspensions or powders to be administered with the aid of conventional devices such as mdis ( metered dose inhalers ), possibly using suitable gaseous carriers such as hfa . the tma doses can be determined by experts in the field on the basis of pharmacodynamic and pharmacokinetic tests , and will also depend on the weight , age and condition of the patient and on other parameters which will be determined by the patient &# 39 ; s doctor . broadly speaking , the effective doses can range from approx . 10 to approx . 500 mg , preferably from approx . 10 to approx . 250 mg of tma a day , possibly divided into a number of administrations , although lower or higher doses cannot be ruled out . the activity of tma as a corrector of f508del - cftr was demonstrated by evaluating the cftr - dependent chloride efflux in polarised cell monolayers , measured by spectrofluorimetry . the use of monoclonal antibodies for cftr has also highlighted the correction of the intracellular localisation , bringing the mutated cftr protein onto the plasma membrane , and the expression of biochemically mature f508del - cftr protein . functional correction of f508del - cftr was assessed by analysing the changes in intracellular cf - dependent mqae fluorescence ( expressed as the f / f 0 ratio ) in human bronchial epithelial cells expressing mutated f508del - cftr protein , grown as polarised cell monolayers on permeable filters , as extensively described previously ( 25 - 26 ). cells were pre - treated or not with the tma or the vx - 809 compounds for 24 hrs , then treated for 3 min with 10 μm fsk plus 100 μm ibmx before substitution of apical chloride by nitrate first in the absence and then in the presence of the specific cftr inhibitor 5 μm cftr inh - 172 ( 27 ). cftr inh - 172 is added apically 5 min before nitrate substitution and remained for the entire efflux . fig1 reports the effect of tma in cfbe41o − polarised monolayers , an immortalised cell line derived from a patient affected by cystic fibrosis carrying the mutated f508del - cftr protein , grown on permeable filters . cells were treated for 24 hrs with increasing concentrations of tma from 50 nm to 500 nm ( grey bars ), or from 500 nm to 5 μm of vx809 ( black bars ). cftr - dependent chloride transport was calculated from the difference in alterations of fsk + ibmx - stimulated fluorescence measurements in the absence and presence of the cftr inhibitor , cftrinh - 172 ( 5 μm ). each bar represents the mean ± s . e . m . for the calculated differences . statistical comparisons were made using an unpaired student &# 39 ; s t test with respect to the values obtained in untreated monolayers ( ctrl ) or in monolayers treated only with the solvent . this example demonstrates that tma is a corrector of the functional defect of mutated f508del - cftr protein in human bronchial epithelial cells . the comparison of tma with the vx - 809 corrector indicates that tma obtains a similar effectiveness but at much lower concentrations than those required for the vx - 809 compound ( 100 nm for tma versus 5 mm for vx - 809 ). the above findings that tma is a functional corrector of the mutated f508del - cftr protein , was checked and extended by assessing the effect of tma on the intracellular localisation of the mutated protein in the same cfbe41o − monolayers at the end of the functional assay described in fig1 , by confocal immunofluorescence microscopy images of polarised cfbe41o − monolayers grown on permeable filters , treated or not with tma ( 100 nm ) or vx809 ( 5 μm ) for 24 hrs . unpermeabilised cells were immunolabelled with a primary mouse monoclonal antibody ( cf3 ) raised against the extracellular first loop of cftr ( 26 ). fig2 shows confocal scans in the vertical cross - section ( xz ) planes . in untreated cells ( ctrl ) or treated with solvent , f508del cftr was not expressed in the apical membrane , whereas after 100 nm tma or 5 μm vx809 treatment , f508del cftr was significantly translocated to the apical membrane ( ap , location of apical membrane ; bl , location of basolateral region . scale bar , 10 μm ). the validation of the effect of f508del cftr protein correctors , such as vx - 809 , before applying the molecules in human clinical trials , is not usually performed in pre - clinical in vivo models , such as in murine strains , but should be carried out by testing more than a single bronchial epithelial cell line expressing the mutated protein in vitro ( 7 , 28 ). tma was therefore tested as a f508del cftr protein corrector in the immortalised cufi - 1 cell line , which derives from a cystic fibrosis patient carrying the mutated f508del cftr protein . expression levels of f508del cftr were analyzed in cufi - 1 cells before and after incubation with tma ( 100 nm ) for 24 hrs by western blotting using anti - hcftr antibody . fig3 shows a representative western blot of a typical experiment . the blot shows that in the cells treated with tma ( 100 nm ), there is an increase in the levels of f508del cftr protein , particularly evident in the mature form of cftr . monolayers of cufi - 1 cells , grown on permeable filters , were treated with tma ( 100 nm ) or with vx809 ( 5 μm ) for 24 hrs and the cftr - dependent chloride transport was determined . the chloride transport was calculated from the difference in alterations of fsk - stimulated fluorescence measurements in the absence and presence of the cftr inhibitor , cftrinh - 172 . each bar represents the mean ± s . e . m . for the calculated differences . statistical comparisons were made using an unpaired student &# 39 ; s t test with respect to the values obtained in untreated monolayers or in monolayers treated only with the solvent . example 3 gives further strong support for the effect of tma as corrector of mutated f508del cftr protein . a further level of pre - clinical validation of mutated f508del cftr protein correctors was obtained by testing the candidate molecule in primary bronchial epithelial cells obtained from the lungs of cystic fibrosis patients without the genetic manipulation inherent in cell immortalisation . these experiments were performed because as it has been demonstrated that f508del cftr protein correctors could be effective in immortalised cell lines but not in the real cellular target , which is most closely represented by the model of the primary bronchial epithelial cells in vitro ( 29 ). the results reported in fig4 show the effect of tma as a corrector in primary bronchial epithelial cells from a cystic fibrosis patient carrying the mutated f508del cftr protein , grown as monolayers on permeable filters in air - liquid interface ( mucilair - cf cells ). primary cells were treated for 24 hrs with 100 nm or 200 nm tma . cftr - dependent chloride transport was calculated from the difference in alterations of fsk + ibmx - stimulated fluorescence measurements in the absence and presence of the cftr inhibitor , cftrinh - 172 ( 5 μm ). each bar represents the mean ± s . e . m . for the calculated differences . statistical comparisons were made using an unpaired student &# 39 ; s t test with respect to the values obtained in untreated monolayers ( ctrl ) or in monolayers treated only with the solvent . after the functional experiments , cells were fixed and immunolabelled with a primary mouse monoclonal antibody ( cf3 ) raised against the extracellular first loop of cftr . confocal immunofluorescence microscopy images of primary mucilair - cf cell monolayers treated or not with tma ( 200 nm ) for 24 hrs are shown . in cells treated with solvent f508del cftr was not expressed in the apical membrane , whereas after 200 nm tma , f508del cftr was significantly translocated to the apical membrane ( ap , location of apical membrane bl , location of basolateral region . scale bar , 10 μm ). the results presented in fig4 strongly confirm that tma is a corrector of the mutated f508del cftr protein non only in immortalised human bronchial epithelial cell lines but also in primary cells derived from the lung of a patient affected by cystic fibrosis . 1 ) welsh j m , ramsey b w , accurso f , cutting g r . cystic fibrosis in “ the metabolic and molecular bases of inherited diseases ”. scriver c r , beaudet a l , sly w s , valle d ( eds ) mcgraw - hill , new york , 2001 . 2 ) jones a m , helm j m . emerging treatments in cystic fibrosis . drugs . 69 : 1903 - 10 , 2009 . 3 ) becq f , mall m a , sheppard d n , conese m , zegarra - moran o . pharmacological therapy for cystic fibrosis : from bench to bedside . j cyst fibros . 10 : s129 - 45 , 2011 . 4 ) van goor f , hadida s , grootenhuis p d , burton b , cao d , neuberger t , turnbull a , singh a , joubran j , hazlewood a , zhou j , mccartney j , arumugam v , decker c , yang j , young c , olson e r , wine j j , frizzell r a , ashlock m , negulescu p . rescue of cf airway epithelial cell function in vitro by a cftr potentiator , vx - 770 . proc natl acad sci usa . 106 : 18825 - 30 , 2009 . 5 ) accurso f j , rowe s m , clancy j p , boyle m p , dunitz j m , durie p r , sagel s d , hornick d b , konstan m w , donaldson s h , moss r b , pilewski j m , rubenstein r c , uluer a z , aitken m l , freedman s d , rose l m , mayer - hamblett n , dong q , zha j , stone a j , olson e r , ordo √± ez cl , campbell p w , ashlock m a , ramsey b w . effect of vx - 770 in persons with cystic fibrosis and the g551d - cftr mutation . n engl j med . 363 : 1991 - 2003 , 2010 . 6 ) flume p a , liou t g , borowitz d s , li h , yen k , ordonez c l , geller d e ; 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