Cystic Fibrosis (CF, OMIM #219700) is an autosomal recessive disorder that commonly affects Caucasians. It is caused by genetic mutations in a gene on human chromosome 7 which codes for an epithelial membrane protein that acts as a cAMP-activated ATP-gated chloride and bicarbonate channel and water transport regulator, known as the cystic fibrosis transmembrane conductance regulator (CFTR) (Rommens et al., 1989). Mutations affecting CFTR gene expression result in difficulties with the processing, folding, or trafficking of the protein to the membrane which adversely affect its function. Among the 2000 different genetic variants causing the disease, the most common is F508del, a deletion of the codon for phenylalanine at position 508. This mutation leads to abnormal folding of the CFTR protein and ultimately, degradation of F508del-CFTR occurs mostly at the proteasome, after ER retention, which prevents its trafficking to the Golgi (Naehrig et al., 2017). Missing CFTR chloride channels at the plasma membrane result in liquid depletion and acidification of the airway surfaces impacting mucus clearance (Ratjen et al., 2015; Veit et al., 2016).

Phenotypically, CF manifestations are extremely variable and can be observed in multiple organs such as the pancreas, the gastrointestinal tract, the reproductive system, and most prominently the lungs (Ratjen et al., 2015; Veit et al., 2016). Moreover, Weber and others demonstrated a link between defective CFTR protein and its implications in the activation of the NF- κ B pathway, associated with the drastic increase of IL-8 (Weber et al., 2001; Rottner et al., 2009).

In addition to cytokines, the impaired inflammatory response in CF reflects an imbalance between the initiation and the resolution of the inflammatory response, with the overexpression of pro-inflammatory cytokines and mediators, while anti-inflammatory and pro-resolving mediators are suppressed. During this imbalance in homeostasis, pro-inflammatory lipid mediators, including 2-series prostaglandins and 4-series leukotrienes, are formed when 20-carbon, omega-6 unsaturated fatty acid, arachidonic acid (AA), is metabolized (Ricciotti and FitzGerald, 2011; Lands, 2015). On the other hand, docosahexaenoic acid (DHA), a 22-carbon unsaturated omega-3 fatty acid, is metabolized to yield anti-inflammatory resolvins and protectins (Serhan et al., 2008), which can orchestrate the timely resolution of inflammation (Kohli and Levy, 2009). Therefore, the elevated AA levels in persons with Cystic Fibrosis (PwCF) directly contribute to an increased production of pro-inflammatory mediators, meanwhile, a decrease in DHA and EPA play important roles in reducing the production of anti-inflammatory mediators (Lachance et al., 2013; Garić et al., 2020b).

It has been well documented that sphingolipid metabolism is dysregulated in CF (Guilbault et al., 2007; Guilbault et al., 2008; Guilbault et al., 2009; Garić et al., 2020b). Ceramides are produced through various pathways and the relative composition of specific species of ceramides depends on the expression of specific combination of ceramide synthases in the organs. In the lungs of healthy mice, very long-chain ceramides (VLCCs, C24:0 CER and C26:0 CER) were shown to represent more than 70% of the total ceramide pool, whereas long-chain ceramides (LCCs, C14:0 and C16:0) constitute less than 20% of the total ceramide pool (Petrache et al., 2013). The relative ratios between VLCCs and LCCs are dramatically affected in CF disease.

In 2006, Fenretinide (FEN), a synthetic retinoid, was reported to effectively inhibit IL-8 release from CFTR-deficient lung epithelial cells (Vilela et al., 2006). Subsequently, FEN has been studied in the context of inflammation in CF disease and its effects have been attributed to its overall ability to trigger the resolution phase of inflammation, as demonstrated by the modulation of macrophage-secreted inflammatory cytokines, to the correction of omega-3/omega-6 fatty acid imbalances impacting phosphorylation of ERK1/2, to the correction of the ceramide deficiency in PwCF, and to the resolution of lung mucus plugging under infection with P. aeruginosa (Lachance et al., 2013; Garić et al., 2020a; Veltman et al., 2021). Furthermore, Garić et al. found a partial synergistic relationship between FEN and Zinc (Zn²⁺), whose deficiency has been reported in PwCF and is associated with severity of CF lung disease (Yadav et al., 2014; Garić et al., 2017).

In 2015, a novel oral formulation of Fenretinide, LAU-7b, was used in a Phase 1, double-blinded, randomized, (3:1, active:placebo), placebo-controlled clinical study, involving 15 adult PwCF (clinicaltrials.gov, NCT02141958). The results from this trial indicated that LAU-7b was safe and well tolerated, while also normalizing lipid imbalances and reducing oxidative stress, as measured in pre-selected plasma biomarkers. Non-clinical experiments with FEN have shown to normalize the aberrant ratio between VLCCs and LCCs as well as AA/DHA ratio in vitro F508del-CFTR expressing cell lines and in vivo (LAU-7b formulation) in mouse models of CF (Lachance et al., 2013; Garić et al., 2020a; Veltman et al., 2021). It is hypothesized that the cumulated benefits of FEN, both during and after the exacerbation episodes, will translate into clinical benefits such as preservation of pulmonary function, reduced incidence and severity of exacerbation episodes, and ultimately a better quality of life for CF patients. This hypothesis is being tested in a Phase II clinical trial with LAU-7b in adult patients with CF (clinicaltrials.gov, NCT03265288).

Several drugs that increase CFTR channel activity, known as potentiators and correctors, have been developed to improve the folding and/or trafficking of the mutated CFTR protein to the plasma membrane. In July 2012, Kalydeco^® (Ivacaftor), the first treatment to specifically target G551D mutations causing CF (4–6% of patients), was approved by the Food and Drug Administration (FDA) in the United States (US). In 2015, it was followed by Orkambi^® (Lumacaftor/Ivacaftor), a combination of CFTR modulators targeting two copies of the F508del mutation (F508del/F508del). In 2018, a more effective combination, Symdeko^® (Tezacaftor/Ivacaftor) addressing a larger number of CF patients (30%) was released. In October 2019, the Triple-combination therapy TRIKAFTA^® was approved in the US for PwCF over the age of 12 with at least one copy of F508del-CFTR and recently has been expanded to include an additional 171 CFTR mutations (Table 3: List of CFTR mutations responsive to VX-445+VX-661+VX-770 (Trikafta®) and approved by the FDA) (Lopes-Pacheco et al., 2021). TRIKAFTA^® is composed of three drugs: Elexacaftor (VX-445), Tezacaftor (VX-661) and Ivacaftor (VX-770) which when used in vitro are referred to as a Triple therapy. Elexacaftor and Tezacaftor are CFTR correctors, which work to increase the amount F508del-CFTR protein that reaches the plasma membrane by aiding its processing and trafficking, while Ivacaftor is a CFTR potentiator that works at the cell surface to increase chloride channel activity (Hoy, 2019).

In this study, we examine whether the combination of LAU-7b with TRIKAFTA^®, could have the potential of translating into a clinical benefit to PwCF.

CF impacts more than 100,000 individuals worldwide (Shteinberg et al., 2021). To date, there is no cure for CF and available treatments target its symptoms. However, CFTR modulator and potentiator therapies which target the genetic defect of the disease, have brought much hope to the CF community. Recently, two randomized, double-blind, phase 3 clinical trials involving TRIKAFTA®, for CF patients 12 years and older, heterozygous or homozygous for the F508del mutation (NCT03525444 and NCT03525548) (Heijerman et al., 2019; Middleton et al., 2019; Zah et al., 2021) were successfully completed and led to the approval of TRIKAFTA® treatment in 2019. More recently, TRIKAFTA® was approved in younger patients, 6–11 years of age, heterozygous or homozygous for the F508del mutation, following a confirmatory Phase 3 trial in this CF population (NCT03691779) (Zemanick et al., 2021). Across all three studies, improvement in percentage of predicted FEV₁ (ppFEV₁) indicated better lung function, lower sweat chloride concentrations and a higher Cystic Fibrosis Questionnaire-Revised respiratory domain (CFQ-R RD) score indicating less respiratory symptoms and a better overall quality of life (Zaher et al., 2021). Furthermore, an observational study in PwCF homozygous for the F508del mutation ranging from 20.8 to 48.3 years old (median age = 31.1 years) taking TRIKAFTA^® over the course of 48 weeks was done (Carnovale et al., 2022). Although inflammation was not evaluated, an improvement in FEV₁, body mass index and sweat chloride was observed. On the other hand, a decrease in exacerbation and a need for intravenous antibiotics was reported. However, it is important to note that due to COVID-19, CF clinics have seen less bacterial exacerbations since patients were isolating and wearing masks. This is relevant given it is a retrospective study (i.e., without adequate control group comparisons) (Carnovale et al., 2022).

While the disease results in the occurrence of many symptoms, lung deterioration caused by repeated bacterial, viral infections, and exacerbations are the main cause of mortality and morbidity (Ratjen et al., 2015). To treat these infections, a combination of anti-bacterial, antifungal, and antiviral medications is needed, but cause complications after repeated and extended periods of use (Hahn et al., 2018). Therefore, novel treatment options that can mitigate the genetic defects of the CFTR protein while also addressing the inflammatory storm seen in PwCF, still need to be explored. Interestingly, a study by Gentzsch et al. found that the rescue of mutant CFTR was improved upon CFTR modulator therapies in vitro undergoing airway epithelial inflammation (Gentzsch et al., 2021). This could prove to be very intriguing and would be interesting to see in an in vivo model.

An interesting strategy, which has gained much ground across various fields in the last decade, is the repurposing and combination of drugs, in the hope of achieving additive and even synergistic effects in treating diseases. In CF disease, small molecule combination has been the strategy employed by the pharmaceutical industries and academic laboratories, which yielded the most successful results (Lopes-Pacheco, 2019; Villella et al., 2019; Pinto et al., 2021).

It was therefore important to investigate whether the inflammation-controlling (pro-resolution) treatment, LAU-7b, would be complementary to the recently approved TRIKAFTA^® therapy and not interfere with the efficacy of the combination in mice with CF lung disease. With the eventual aim of improving the quality of life for PwCF and to address a broader range of the pathology observed in CF lung disease, we have assessed the efficacy of the combination of pro-resolution LAU-7b, with modulator and corrector therapies.

This study examined the impacts that combinatory treatment of LAU-7b (oral fenretinide) and a Triple therapy similar to TRIKAFTA^® have in vivo on DD mice, and also in vitro, using a CFBE overexpressing F508del cell line. While each treatment on its own has demonstrated ameliorative effects in the studies conducted thus far, their combination is studied here for the first time. TRIKAFTA^®’s mechanism of action remains to be fully elucidated. However, it is thought that correctors, Elexacaftor and Tezacaftor, directly interact with the mutant CFTR protein, facilitating its movement and trafficking to the Golgi, while the potentiator Ivacaftor, increases channel activity at the membrane level (Zaher et al., 2021). LAU-7b′s mechanism of action was previously discussed; functioning as a membrane lipid modulator and exerting inflammation-controlling (pro-resolution) in multiple in vitro and in vivo systems (Garić et al., 2017; Garić et al., 2020b). Given our understanding of the two treatments and the benefits they each have, we began by studying their combination in vivo in our older, 14 to 20-month-old DD and WT mice. Before studying the effect of various infections on the efficacy of TRIKAFTA^®, it was important to first study mice that develop CF lung disease even under pathogen-free conditions. This occurs when CF mice reach an age corresponding to that of PwCF at which those with a class 1 and 2 mutations in the CFTR gene have a decline in the force expiratory volume in 1 s (FEV₁).

After diagnosis of CF lung disease, a standard measure of disease progression and airway obstruction is FEV₁ tested by spirometry (Szczesniak et al., 2017). Declining pulmonary function among PwCF is age-dependent, however, the most dramatic drop in FEV₁ occurs between puberty and the age of 30, although it is dependent on the CFTR gene mutation. The decline in lung function over time in CF patients results from chronic airway inflammation and mucus dehydration, which are triggered by infection-induced increases in pro-inflammatory lipid mediators, which are regulated by AA release (Miele et al., 1997; Elizu et al., 2008). In mice, an invasive measure of airway resistance is a gold standard assay performed to monitor pulmonary function. In Figure 1A, our WT control group showed an expected slight increase upon MCh addition. However, a significant impairment in airway function is seen for the DD NT and VEH treated mice, most notably at the 100 mg/ml MCh dose, upon comparison to WT control (Figure 1B). Moreover, a significant improvement is seen upon treatment with LAU-7b, which shows airway resistance levels comparable to healthy WT controls. While Triple therapy treatment improves airway function, its effects are less marked than those seen in LAU-7b treated animals. The mice treated with the combination of Triple therapy and LAU-7b, displayed a significant improvement in lung function. The normalization of lung function upon combinatory treatment denotes a lower airway hyperresponsiveness, like that of WT control mice. This indicates that mice treated with LAU-7b, or LAU-7b + Triple therapy, have significantly better pulmonary function than mice treated with only Triple therapy.

In PwCF, the abnormal mucosal defences facilitate recurrent chronic infection, most often with P. aeruginosa and sometimes with Aspergillus fumigatus, which negatively impacts the inflammatory milieu leading to lung damage and eventually lung failure, the main cause of morbidity and mortality in PwCF (Leclair and Hogan, 2010; Ratjen et al., 2015; Cottrill et al., 2020). It is well documented that MUC5AC overexpression plays a key role in airway plugging, and both MUC5AC and MUC5B are not only upregulated in chronically infected PwCF but are further augmented during lung exacerbations that happen in a large percentage of PwCF 3–4 times a year (Rowe et al., 2005; Fahy and Dickey, 2010; Garić et al., 2020a). To improve mucus clearance, mucoactive agents, consisting of mucolytics and hyperosmolar agents, can be used (Hurt and Bilton, 2014). Bronchodilators work to improve airway opening by relaxing the smooth muscles in the bronchial wall facilitating mucus clearance (Smith and Edwards, 2017; Smith et al., 2020). Despite various treatments, delayed resolution of inflammation that frequently occurs in PwCF following exacerbation, often results in permanent lung damage. To assess lung deterioration in our F508del mice that were kept in a specific pathogen free condition (SPF), histological analysis of lung sections stained with H&E was done (Figure 3). Histology revealed overall ameliorated pathological parameters in mice treated with LAU-7b and LAU-7b in combination with Triple therapy as compared to vehicle treated controls, further supporting the results obtained for the physiological assessment. Hyperplasia of airways with significantly thickened airway lining is a pathological feature of cystic fibrosis and other respiratory diseases (Gorrieri et al., 2016). In Figure 3, a major difference can be seen between the airways of the WT control mouse and the DD-NT or DD-VEH mouse. Both DD-NT and DD-VEH mice have elevated levels of hyperplasia compared to WT control. These results corroborate the findings of a lower airway resistance in WT mice, and higher resistance in DD NT and VEH treated mice, highlighting the drastic difference in overall pulmonary health for these two groups. While treatment with LAU-7b or Triple therapy reduce airway cell hyperplasia, the combination of LAU-7b and Triple therapy yields superior results compared to Triple therapy alone, with uniform looking lungs, comparable to those of WT controls. The histology images are further supported by Figure 3G, displaying the lowest number of infiltrating cells for the combinatory treatment group.

In response to allergic reactions or infections, goblet cells present in the airways start production of mucus, and an increase in the reproduction rate of these cells leads to mucus hypersecretion, leading to abnormal mucus accumulation and airway plugging. This results in decreased antimicrobial functions and impaired mucociliary clearance, which can further worsen the CF condition (Puchelle et al., 2002). Histological assessment of lung sections demonstrates that treatment with LAU-7b and Triple therapy dramatically diminish the thickening of the airway lining. As the inflammatory response in the lungs of PwCF is self-perpetuating and can be worsened by abnormal immune activity, it is important that any developing CF treatment considers the likely possibility of goblet cell hyperplasia and its consequences. Mucin expression that is inducible upon allergic response to Aspergillus antigens and infections with P. aeruginosa, Staphylcoccus aureus and other bacterial and fungal induced lung pathology was not evaluated in the current study, but is one of the important aspects of our ongoing studies.

Since oxidative markers, fatty acids, and overall lipid profiles, are known to be dysregulated in both CF mice and PwCF from birth, prior to their development of CF lung disease (Strandvik et al., 2001; Strandvik, 2010; Youssef et al., 2020), further analysis was done using a portion of the liver, lung and plasma of WT and DD mice under the various treatments to obtain the lipidomic profile of those mice, first, oxidative stress markers were analyzed. Being a universal biological response, oxidative stress plays a major role in a variety of inflammatory disease conditions. In CF, neutrophils are continuously recruited to the airways and liberate their toxic products such as oxidants, in an uncontrolled fashion (Hector et al., 2014). While antioxidants shield the lung from free oxidative damage in healthy patients, the amount and duration of neutrophilic inflammation overwhelms these defence systems in CF subjects, leading to increased protein and lipid oxidation in the lungs. To improve the defense system in PwCF, it is important to introduce a treatment which reduces levels of lipid and protein oxidation as marked by MD and 3-NT, respectively. In Figure 4, LAU-7b treatment in DD mice significantly reduced levels of MD and 3-NT in the lungs, plasma, and liver, to a greater extent than Triple therapy treatment. However, upon combination with Triple therapy, improvements are comparable to that of LAU-7b alone, lowering the levels of those oxidative markers below the WT threshold. Thus, the combination of Triple therapy with LAU-7b, is significantly better than Triple therapy alone, given LAU-7b′s antioxidant properties counteracting the overwhelming recruitment of neutrophils seen in lung inflammation.

Moreover, fatty acids abnormalities, in AA, DHA, and EPA, are consistently reported in CF (Sinaasappel et al., 2002). These abnormalities create an imbalance in the DHA/AA ratio in favour of AA, contributing to the increase in pulmonary inflammation and mucus dehydration, resulting in deterioration of PwCF’s condition. However, the causal connection between the expression of CFTR protein and this phenomenon has rarely been examined. Some evidence that suggests a role for CFTR in fatty acid metabolism as it was shown in cell culture models, where CFTR dysfunction results in defective fatty acid composition (Andersson et al., 2008). Furthermore, similar polyunsaturated fatty acid changes in CF affected organs such as the lung, pancreas and ileum have been reported in CFTR knock-out mice, further suggesting a causal link between CFTR and fatty acid metabolism (Freedman et al., 1999). Further studies also report that modulation of saturated fatty acids correlates with the modulation of LCCs and VLCCs (Garić et al., 2020a). In Figure 5, EPA and DHA levels are shown in the lungs, plasma and liver of DD mice. Upon treatment with LAU-7b, a significant increase in those omega-3 fatty acids was observed. While treatment with Triple therapy alone did show some improvement, it is only after its combination with LAU-7b that we see an increase in EPA and DHA comparable and even exceeding levels of EPA and DHA seen in WT mice. Combinatory treatment with Triple therapy and LAU-7b shows normalization of AA levels, which is not seen with Triple therapy treatment alone. Figures 6 and 7 show LCCs and VLCCs levels in lung, liver and plasma, respectively. In 2020, Liessi et al. performed an untargeted lipidomic analysis on CFBE41o-cells upon various treatment groups, which included the Triple combination therapy (VX-661/VX-445/VX-770) (Liessi et al., 2020). Our results, as seen in Figures 6 and 7 corroborate their findings (Figure 4), of a downregulation in C14:0 and C16:0 and a concomitant upregulation in VLCCs (Liessi et al., 2020). Recently, an interesting study was published by Westholter and others reporting ceramide levels obtained from plasma analysis of 25 PwCF (age 35.56 ± 12.75; 20 out of 25 with intermittent or chronic Pseudomonas infection) treated for 4 weeks with TRIKAFTA® (Westhölter et al., 2022). A very modest improvement (decrease) in C16:0 ceramide levels (0.218 ± 0.09 before treatment and 0.178 ± 0.06 (p = 0.0051) after 4 weeks of treatment) with TRIKAFTA® was reached. Furthermore, a very modest improvement in the levels of C24:0 (1.354 ± 0.47 before and 1.674 ± 0.65 after 4 weeks of treatment (p = 0.0048)) was also achieved. However, only 3 PwCF out of 25 reached levels for C24:0 of 2.5 after treatment, with none of the patients reaching a level of 4, which is typical for healthy individuals (Garić et al., 2017). No improvement in C22:0 or C24:1 ceramide was reported following the treatment with TRIKAFTA® and no analysis of C26:0 ceramides was done. The ratio between C16Cer/C24Cer has improved from 0.171 ± 0.06 before treatment to 0.112 ± 0.03 after 4 weeks of TRIKAFTA® treatment (p-value = <0.001) (Westhölter et al., 2022).

In Figure 8 the VLCC/LCC ratio, which is known to be altered in CF disease, is emphasized. While Triple therapy can partially improve this ratio by increasing VLCC levels and decreasing LCC levels, the greatest correction occurs upon combinatory treatment with LAU-7b, surpassing the WT threshold in lungs, liver, and plasma of WT mice. Overall, Triple therapy alone seems to be beneficial in terms of correcting the pool of fatty acids, and ceramides, however, the combination of Triple and LAU-7b is demonstrated to be superior in further ameliorating these levels to similarity with what is seen in WT mice. This cooperative effect might be particularly important for PwCF since most of them are chronically infected with bacteria such as P. aeruginosa, and/or fungi.

Results obtained in vitro corroborate the in vivo findings, once again demonstrating that the combination of Triple with LAU-7b is more efficacious in improving the VLCC/LCC ratio of the CFBE41o-(F508del) cell line, than Triple therapy alone. Interestingly, our data demonstrated that fenretinide treatment increases total CFTR protein levels (Figure 9), in the same cell line, thereby providing more protein that can be subsequently processed to the cell membrane with the help of modulators and the potentiator, enabling functional recovery of CFTR channel.

Ultimately, there is a benefit for PwCF from TRIKAFTA®. Moving forward, it is imperative to study whether the effect of TRIKAFTA® is long-lasting and whether the lung inflammation resulting from recurrent lung infections does not diminish its efficacy over time, as previously seen with Kayldeco®, a similarly effective CFTR modulator (Heltshe et al., 2018). The combination of Triple therapy and LAU-7b might resolve this important facet. Taken together, the results obtained in this study strongly suggest for a potential clinical benefit from using TRIKAFTA® in combination with LAU-7b, with the aim of slowing the lung degradation and further improving overall quality of life in PwCF.

While the study presented above can have a lasting impact for PwCF, there are limitations. The cost of the Triple treatment was a limiting factor in our decision for the number of mice to include in each treatment group. Therefore, we chose to include 3 mice in each Triple and Triple + LAU-7b groups, given it is the minimum number of mice required to achieve statistical significance. Furthermore, kinetics of inflammatory mediators and mucin induction, which is usually following exposure to lung pathogens or their filtrates, could not be evaluated in this study since animals were maintained in a specific pathogen-free (SPF) condition. The airway thickening quantification in Figure 3G is based on a semi-quantitative assessment of four airways per mouse lung at ×20 magnification and establishment of a fully quantitative approach would be desirable in the future.