CFTR channel activator compounds and pharmaceutical...

Details CFTR channel activator compounds and pharmaceutical... CFTR channel activator compounds and pharmaceutical...

1999-03-02

2003-10-07

Aulakh, Charanjit S. (Department: 1612)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C546S095000, C546S071000, C514S284000

Reexamination Certificate

active

06630482

ABSTRACT:

This application is a 371 of PCT/FR97/01436 filed Jul. 31, 1997, now WO 98/05642 Feb. 12, 1998.
The present invention relates to CFTR channel activator compounds, pharmaceutical compositions comprising the latter, and their use in the context of treatment of pathologies such as cystic fibrosis.
In an epithelial cell, transportation of water and electrolytes is associated with an increase in the permeability of the membranes to the ions K
+
, Na
+
and Cl
−
. These movements are linked to the activity of ion channels, that is to say specialized proteins integrated into the membrane allowing passive diffusion of ions. The techniques of molecular electrophysiology (patch clamping) allow recording at the unit level of the openings and closings of an ion channel and make it possible to study transepithelial ion transportations their regulation and their pathological dysregulation.
Among the numerous pathologies associated with the physiology of epithelial cells, cystic fibrosis is also regarded as a pathology of ion channels to the extent that the protein involved is a chloride channel, the CFTR channel, meaning “cystic fibrosis transmembrane conductance regulator”. Mucoviscidosis, or cvstic fibrosis (CF) in Anglo-Saxon terminology, is the most common recessive autosomal genetic disease in Caucasian populations. In the United States and in the majority of European countries, the incidence of carriers of the CF gene is 1 in 20 to 1 in 30. Cystic fibrosis affects the exocrine glands of the human organism. The main expression sites of the CFTR protein are the exocrine pancreas, the lungs, the sudoriparous glands, the intestine and the cardiac tissue. The attention paid to this disease has had significant consequences on understanding the secretory mechanisms of normal epithelial cells. The epithelial cells of the exocrine glands of the intestine, the pancreas or the lungs control transportation of salt and water into these organs. In cases of cystic fibrosis, mutations of the CF gene alter the properties and function of the CFTR channel. The transportation of electrolytes then becomes abnormal and leads to chronic pulmonary obstructive disorders, to pancreatic insufficiency, to bacterial pulmonary conditions, to an abnormally concentrated sudoriparous secretion and to masculine infertility. The defective secretion is linked with functioning of selective ion channels for chloride ions (CFTR channels) which are located in the apical membrane of cells and the activity of which is controlled by the cyclic AMP route.
The protein CFTR is a glycoprotein of 1.480 amino acids of molecular weight 170 kD divided into five fields (Riordan et al. 1989), two transmembrane segments each with 6 alpha-helices (numbered 1 to 12, each comprising 21 to 22 amino acids), two nucleotide binding fields (NBF1 and 2) and a large hydrophilic regulation field (field R). The protein CFTR, from its molecular structure, belongs to the family of membrane transporters (ABC meaning ATP-binding cassette).
The ABC transporters constitute a family of membrane proteins which are highly conserved in evolution. They are involved in the translocation of various substrates through cell membranes. However, while in prokaryotes several transporter/substrate pairs have been defined, this information is rarer in eukaryotes. In mammals, the majority of ABS transporters are associated with a pathology. The protein CFTR involved in cystic fibrosis, glycoprotein P (MDR: multi-drug resistance) involved in the rejection of antitumoral cytotoxic drugs and the protein ABC 1, recently described as playing an essential role in endocytosis of apoptotic bodies by the macrophage, may be mentioned. CFTR controls the transportation of transepithelial chloride and hydration of mucous compartments, while one of the isoforms of MDR is involved in the translocation of phosphatidylcholine. These three ABC proteins, which have a structure of two times 6 transmembrane segments, have two fields which bind and hydrolyse nucleotides (NBF) and a regulator field. The regulation of CFTR has been studied in particular.
Two complex processes control the activity of the CFTR channel: phosphorylation of field R by kinase proteins and binding (and perhaps hydrolysis) of ATP on the two NBF fields. The dephosphorylation of the CFTR channel causes a loss in activity of the channel up to its closure (Tabcharani et al., 1991, Becq et al. 1993a. Becq et al., 1994). In addition, the CFTR channel is associated with a membrane phosphatase which controls the activity and state of phosphorylation of the channel (Becq et al., 1993b. Becq et al., 1994).
The gene which codes for the protein CFTR has been isolated by molecular cloning and identified on chromosome 7 (Kerem et al., 1989, Riordan et al., 1989). The identification of the gene and its involvement in cystic fibrosis has been confirmed by the location of a deletion of three base pairs in a coding region (exon 10) of the CF gene originating from CF patients. This mutation corresponds to the deletion of a phenylalanine in position 508 (&Dgr;F508) of the protein in NBF1. The frequency of occurrence of this mutation is 70% on average in the gene analyses (Tsui & Buchwald. 1991). The consequences of this mutation are dramatic, since the abnormal protein produced by transcription of the mutated gene (&Dgr;F508) is no longer capable of ensuring its function in the transportation of chloride of the epithelial cells affected. The absence of a chlorine current after stimulation of epithelial cells of exocrine glands by cAMP is the main characteristic demonstrating the presence of an anomaly of the CF gene, and in particular the mutation (&Dgr;F508). More than 300 mutations have been identified to date on the CF gene. The highest density of mutations is found in the two nucleotide binding fields. The mutation (&Dgr;F508) is found in 70% of cases, and 50% of patients are homozygous for this mutation. Seven other significant mutations are present with incidences of greater than 1%. The mutation G551D corresponds to replacement of a glycine residue (G) in position 551 of the protein by an aspartic acid (D). Patients who carry this mutant have a severe pathology with a pancreatic insufficiency and serious pulmonary disorders (Cutting et al., 1990). The incidence of observation of this mutation reaches 3 to 5% in certain CF populations. In contrast to the &Dgr;F508 deletion, the protein CFTR carrying the mutation G551D is mature and is incorporated into the membrane (Gregor et al., 1991). However, the mutation causes an impermeability of the membrane and stimulation of the cAMP route does not open the channel associated with expression of this mutant (Gregory, et al., 1991. Becq et al., 1994).
Other mutations, such as R117H, R334W and R347P, appear with low incidences of 0.8, 0.4 and 0.5% respectively, and are associated with a less serious pathology (Sheppard et al., 1993). Expression of these three mutants generates a mature glycosylated form of the protein in harmony with its insertion into the membrane.
However, the three mutants are capable of responding to stimulation of the AMP route by opening of the channels. The amplitude of the currents, the unit conductance and the probability of opening of the channel associated with each of the three mutants are modified with respect to the normal CFTR channel (Sheppard et al., 1993. Becq et al., 1994). However, regulation by kinases/phosphatases seems normal for these various mutants, including the mutants G551D and &Dgr;F508 (Becq et al., 1994).
These observations thus show that it is possible pharmacologically to activate a large number of CFTR mutants, including G551D and &Dgr;F508. In spite a lack of directing of the protein &Dgr;F508 in the membranes of epithelial cells affected by cystic fibrosis, several teams have shown that this protein could be present in a functional manner in the membranes (Dalemans et al., 1991, Drumm et al., 1991. Becq et al., 1994). It therefore seems necessary and of primary importance to develop a strategy for opening CFTR channels to

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