**3. Conclusions**

The clinical use of these molecules may be possible after further studies relative to the shortand long-term effects of potent NMD inhibition as the combination of a read-through approach with pharmacologic NMD inhibition is an appealing therapeutic strategy for CF patients

Clancy's group [16] nasally administered aminoglycosides did not produce detectable changes in CFTR function in subjects heterozygous for a variety of stop codon mutations within CFTR, suggesting the need to perform a combined therapy based on the most potent suppressors of nonsense mutations co-delivered with CFTR potentiators and/or correctors [16]. Many CF patients heterozygous for premature stop mutations carry a genotype with F508del. Initial results of a Phase II trial of VX-809, a lead F508del CFTR corrector developed by Vertex Pharmaceuticals (Cambridge, Massachusetts, USA), established that systemic administration of the compound for 4 weeks modestly improved sweat chloride at the highest dose tested compared with placebo [44]. VX-809 is reported to normalize the gating of corrected F508del-CFTR but has no direct potentiating action suggesting that one straightforward approach could be the co-administration with a potentiator of CFTR channel gating [11,44,45]. If CF patients have nonsense mutations together with F508del, a cocktail of potentiators of CFTR channel gating, CFTR correctors, and read-through molecules could be proposed as a therapeutic

Xue X et al. [22] reported a novel synthetic aminoglycoside based on NB124 that efficiently restored CFTR function in primary human bronchial epithelial (HBE) CF cells carrying PTC (G542X/delF508). The efficacy of NB124 was further enhanced by addition of the oral CFTR potentiator Ivacaftor (VX-770) to airway cells expressing CFTR PTCs [22]. Ivacaftor exhibited excellent activity and pharmacokinetic properties, providing the opportunity to treat the underlying cause of CF in combination with CFTR correctors and read-through molecules,

Many new compounds are expected to be proposed in the future for the potentiation and correction of CFTR. One example is 4,6,4'-trimethylangelicin (TMA), a psoralen-related compound, which obtained the orphan drug designation from the EMA and is already in clinical use for psoriasis. Tamanini et al. [47] demonstrated that TMA, at nanomolar concen‐ trations, inhibited the expression of the IL-8 gene in bronchial epithelial cells in which the inflammatory response has been challenged with *P. aeruginosa*. The acute addition (15 minutes treatment) of 250 nM TMA potentiated FSK-stimulated chloride secretion in airway cell monolayers expressing wild type CFTR or in CF cells in which F508del CFTR was already rescued to the apical membrane by overexpressing the interacting protein NHERF1. More recently, the same group [48] found that long pre-incubation with nanomolar concentrations of TMA was able to effectively restore both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. These results indicate that TMA, besides its anti-inflam‐ matory and potentiator activities, also displays corrector properties, suggesting that this

**2.3. Treatment of CF heterozygous patients for nonsense mutations**

carrying nonsense mutations.

316 Cystic Fibrosis in the Light of New Research

strategy.

depending on the CFTR mutations [46].

More than 1,900 different CFTR mutations have been reported in CF patients, 10% being nonsense mutations and 75% represented by the most frequent CF F508del mutation [1,11]. In order to ameliorate the CF phenotype, restoration of at least 5–35% of normal CFTR function is required [2-4]. Several CFTR modulator therapies have been investigated in vitro and in late phase clinical trials, including CFTR potentiators, correctors, and PTC read-through molecules [1,2-5,9-16,21-30,37-39,42,45]. To achieve substantial clinical benefit in CF, a combined admin‐ istration of two correctors with synergistic action [55,56] or a corrector with a potentiator that counteracts distinct conformational defects in trafficking and activation of F508del CFTR [57, 58] are required.

Current open clinical trials, excluding studies with unknown status, are being conducted on Ivacaftor and PTC124 (Table 2).

Ivacaftor alone is not sufficient to efficiently alter CFTR activity in patients with CF homozy‐ gous for F508del [59]. The difficulty in achieving higher levels of correction is due to the fact that the F508del mutation induces multiple conformational defects in the mutant protein.

In current clinical trials (Table 2) Ivacaftor is being tested in combination with CFTR correctors such as Lumacaftor (VX-809, Vertex Pharmaceuticals, Cambridge, Massachusetts, USA) and VX-661 (Vertex Pharmaceuticals) in order to correct the protein misfolding and increase the CFTR localization to the cell surface.

VX-809 is selective for F508del CFTR and enhances chloride secretion to 15% of that found in non-CF human bronchial cells [45,48,55]. Preclinical research [45,60] and known effects of the F508 deletion on gating in addition to cellular processing [56-58] support the combination strategy of VX-809 (or VX-661, an alternate CFTR corrector to VX-809) with Ivacaftor in current human clinical trials (Table 2).

Alternatively, dual-acting molecules (such as TMA [47,48], aminoarylthiazole [61], and cyanoquinoline [49,62] derivatives) that both correct F508del CFTR and potentiate the F508del CFTR-dependent chloride permeability has been proposed, avoiding multiple combined administration.


**Table 2.** Current open clinical trials on CFTR modulators (source ClinicalTrials.gov).

Other ongoing human clinical trials are investigating novel CFTR modulators on F508del such as N91115, glycerol phenylbutyrate, FDL169 (Table 2). N91115 is currently in Phase I. In next Phase II this molecule will be studied in addition to Lumacaftor/Ivacaftor.

The combination of CFTR potentiators and correctors could result in effective therapies in a large proportion of patients with CF (Table 2).

Other CFTR modulator classes include molecules with read-through activity, such as PTC124 currently in Phase III clinical study. Based on efficacy in vitro and in animal models [10,25,63], PTC124 was studied in a series of conflicting Phase II trials [26-28,63]. A long-term study has demonstrated no improvement in FEV1 % predicted, the primary endpoint. Conversely a small effect on lung function was demonstrated in a predefined subset of individuals untreated with inhaled antibiotics that can modify the efficiency of PTC124 [29,63]. So further clinical studies for this compound should be necessary to demonstrate its activity in CF as read-through therapeutic approach also in combination with NMD inhibitors or potentiators/correctors as reported in studies in vitro [22,43].

Results to date suggest, in the case of nonsense mutations, efficient and therapeutic restoration of a functional CFTR channel may be possible drugs administered in combination.

In conclusion, it is essential to stratify CF patients with the aim of creating a personalized therapy based on the use of a combination of drugs targeted at specific classes of mutations. This approach represents an appealing therapeutic strategy and should be the subject of further investigation.
