Lumacaftor/ivacaftor improves liver cholesterol metabolism but does not influence hypocholesterolemia in patients with cystic fibrosis
Monica Gelzo a,b, Paola Iacotucci c, Mafalda Caputo a, Gustavo Cernera a,b,
Marika Comegna a,b, Vincenzo Carnovale c, Gaetano Corso d,∗, Giuseppe Castaldo a,b
a Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy
b CEINGE - Biotecnologie avanzate, Naples, Italy
c Dipartimento di Scienze Mediche Traslazionali, Università di Napoli Federico II, Naples, Italy
d Dipartimento di Medicina Clinica e Sperimentale, Università di Foggia, Foggia, Italy
a r t i c l e i n f o
Received 26 November 2019 Revised 17 June 2020 Accepted 17 June 2020 Available online xxx
Cholesterol homeostasis Cystic fibrosis
Gas chromatography Lumacaftor/ivacaftor Pancreatic insufficiency
a b s t r a c t
Background: Cystic fibrosis (CF) patients have reduced intestinal absorption of sterols and, despite en- hanced endogenous synthesis, low plasma cholesterol. Lumacaftor/ivacaftor CFTR protein modulator ther- apy is used to improve the clinical outcome of CF patients homozygous for F508del mutation (homo- deltaF508). Aim of the study is to evaluate the cholesterol metabolism and hepatobiliary injury/function in adult homo-deltaF508 patients, before and after lumacaftor/ivacaftor treatment. Baseline parameters in homo-deltaF508 patients were compared to those in CF patients compound heterozygous for F508del mutation and another severe mutation (hetero-deltaF508).
Methods: Cholesterol metabolism was evaluated measuring plasma phytosterols and cholestanol, as in- testinal absorption markers, and lathosterol, as liver biosynthesis marker. We quantified serum vitamin E, as nutritional marker. We evaluated liver injury by aspartate aminotransferase (AST) and alanine transam- inase (ALT), biliary injury by γ -glutamyltransferase (γ GT) and AP, and the liver function by bilirubin and albumin.
Results: Before the treatment, homo-deltaF508 patients (n = 20) had significantly lower cholesterol and vitamin E compared to hetero-deltaF508 (n = 20). Lumacaftor/ivacaftor treatment caused: 1) further re- duction of cholesterol; 2) lathosterol reduction, suggesting a normalization of endogenous synthesis; 3) cholestanol and vitamin E increment, indicating an improvement of lipid digestion/absorption. Vitamin E difference (after-before treatment) was positively associated to treatment months. Alkaline phosphatase was also reduced.
Conclusions: These data suggest an effect of lumacaftor/ivacaftor on cholesterol metabolism and entero- hepatic flux in CF patients. However, lumacaftor/ivacaftor does not promote the increase of cholesterol serum concentration that on the contrary declines. Further studies are needed to research the real mech- anism causing this reduction.
© 2020 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
Abbreviations: ALT, alanine transaminase; AP, alkaline phosphatase; AST, as- partate aminotransferase; CF, cystic fibrosis; CFLD, CF-associated liver disease; CFRD, CF-related diabetes; CFTR, cystic fibrosis transmembrane regulator; γ GT, γ -glutamyltransferase; hetero-deltaF508, CF patients compound heterozygous for F508del mutation and another severe mutation; homo-deltaF508 patients, CF pa- tients homozygous for the F508del mutation; FEV1, forced expiratory volume in the 1st second; IQR, interquartile range; PI, pancreatic insufficiency.
∗ Corresponding author.
E-mail address: [email protected] (G. Corso).
Among a myriad of clinical alterations, most patients with cys- tic fibrosis (CF) have low plasma cholesterol due to intestinal mal- absorption, despite the pancreatic enzyme supplementation [1,2]. In particular, we previously found that CF patients with pancreatic insufficiency (PI) have reduced plasma phytosterols levels, markers of intestinal absorption of cholesterol, that were positively corre- lated with vitamin E . The serum level of vitamin E represents an indirect pancreatic function test that allows to evaluate the in- testinal digestion/absorption of lipids . On the other hand, an enhanced liver biosynthesis of cholesterol was demonstrated by
1569-1993/© 2020 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
Please cite this article as: M. Gelzo, P. Iacotucci and M. Caputo et al., Lumacaftor/ivacaftor improves liver cholesterol metabolism but does not influence hypocholesterolemia in patients with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2020.06.015
deuterium incorporation in CF cells and animal models [4,5] as well as in CF patients by elevated plasma levels of a surrogate marker of synthesis, i.e., plasma lathosterol . The enhanced liver biosynthesis is unable to maintain normal plasma cholesterol lev- els, likely because the altered CF transmembrane regulator (CFTR) activity impairs liver cholesterol secretion [4,5].
Recently, molecular drugs became available for patients with CF bearing specific genotypes, among which potentiators  that improve the CFTR gating, and correctors  that act as chap- eron for CFTR protein mislocalization. More recently, the combi- nation of the potentiator Ivacaftor and the corrector Lumacaftor became available to treat CF patients older than 6 years homozy- gous for the F508del mutation . The novel therapy was safe and showed slight but significant improvements of respiratory func- tion (predicted FEV1) and a 35% reduction of pulmonary exacerba- tions . A prospective observational study on 53 patients with CF treated with lumacaftor/ivacaftor demonstrated an improvement of FEV1 and BMI and a significant reduction of sweat chloride and nasal potential difference and the improvement of intestinal cur- rent measurements. However, no correlations were found between these parameters and the clinical outcome . Thus, a phase III study on 58 patients (age 6–11 years) revealed a modest but clin- ically significant improvement in lung function, a slower decline in FEV1 and a reduction of pulmonary exacerbations. These im- provements were maintained in a two-year open label follow up . However, the study reported a high occurrence of altered liver function and severe adverse liver-related reactions, particularly in patients with preexisting liver disease. Moreover, an isolated ele- vation of alkaline phosphatase (AP) in association with lumacaftor- ivacaftor treatment has been reported .
Until now, none of these studies explored the effects of lumacaftor/ivacaftor on cholesterol metabolism. Thus, in the present study we evaluated the cholesterol metabolism and hepa- tobiliary injury/function in adult patients with CF homozygous for the F508del mutation (homo-deltaF508), before and after at least 9 months of treatment with lumacaftor/ivacaftor, and in untreated CF patients, that were compound heterozygous for F508del muta- tion and another severe mutation (hetero-deltaF508).
To this purpose, we evaluated cholesterol metabolism by the analysis of plasma surrogate markers, i.e., lathosterol, phytosterols and cholestanol. The latter is also a marker of intestinal absorption as well as, in some metabolic diseases, is a secondary marker of cytochrome P450 27A1 defect (i.e., cerebrotendinous xanthomato- sis) [1,12]. The serum lipid profile together with vitamin E lev- els were also analyzed. We evaluated the liver disease by clin- ical and laboratory markers such as aspartate aminotransferase (AST) and alanine transaminase (ALT) for hepatocytes injury, γ - glutamyltransferase (γ GT) and AP for biliary injury, and bilirubin and albumin for liver function .
- Patients and methods
In this study we recruited 20 homo-deltaF508 patients, who were starting the lumacaftor/ivacaftor treatment (2 × 200/125
tabs q 12 h) at the Regional Cystic Fibrosis Reference Center for adults (Department of Translational Medical Sciences, University of Naples Federico II). The treatment was approved by the Ital- ian Medicines Agency (AIFA) to treat the underlying cause of Cys- tic Fibrosis in people ages 12 and older with two copies of CFTR gene with F508del mutation. In addition, we recruited 20 un- treated hetero-deltaF508 patients, matched for age and sex to homo-deltaF508 patients. The CFTR genotype of all patients was defined by the first level molecular analysis  and in all cases gene sequencing excluded other in cis mutations [15,16]. All in-
cluded patients had PI and all of them were already treated with pancreatic enzyme replacement therapy (PERT). All patients re- ceived multivitamin supplementation (2 tabs of DKX, Neupharma, Italy) containing vitamin E (362 mg/day), except for two untreated patients that received 1 tab of Kledax (Chiesi Farmaceutici SPA, Italy; vitamin E: 267 mg/day).
The study was performed according to the current version of the Helsinki Declaration and all subjects were informed and gave written permission to process anonymously their clinical results for scientific aims.
Plasma and serum samples were collected from fasted patients. For homo-deltaF508 patients we collected the samples before and after lumacaftor/ivacaftor treatment. The period of treatment (from 2017 to 2019) ranged from 9 to 29 months (mean (SD): 15 (6) months).
Serum samples were separated from blood cells and then an- alyzed for biochemical parameters by an automated biochemistry analyzer (Architect ci 16,200 Integrated System, Abbott Diagnostics, Rome, Italy) as previously described [17,18]. All procedures were under quality control assurance internal and external laboratory. Serum vitamin E was analyzed by an isocratic high performance liquid chromatography (HPLC) method (Vitamin A/E by HPLC assay, Bio-Rad Laboratories, Segrate, Italy) with an HPLC-UV system (Ag- ilent 1260 Infinity Quaternary LC coupled with BIO-RAD UV-1806, Bio- Rad Laboratories, Segrate, Italy).
Plasma samples were separated from blood cells after the col- lection and stored at -20 °C after the addition of butylated hy-
droxytoluene, as antioxidant [19,20]. The analysis of plasma sterols was performed on 50 μL of sample by standardized gas chromato- graphic method as previously described [1,21].
Continuous data were reported as mean (SD) for normal dis- tributions or median (interquartile range, IQR) for non-parametric distributions. Shapiro-Wilk test was used to test the normality of distributions. Categorical data were reported as frequency and per- centage. Comparisons between two groups with normal or non- parametric distributions were performed by unpaired t-test or Mann-Whitney U test, respectively. Comparisons of paired data were performed by paired t-test or Wilcoxon signed-rank test as appropriate. The chi-square test was used to compare the fre- quency of categorical variables between groups. The associations between variables with normal distributions were evaluated by Pearson r correlation. The significance was accepted at the level of p < 0.05.
Table 1 describes demographic, anthropometric, genetic and clinical data of homo-deltaF508 patients before the treatment
(n = 20) in comparison with hetero-deltaF508 patients (n = 20). Both groups of CF patients were PI and received comparable PERT and vitamin E supplementation. The frequencies of patients with CF-associated liver disease (CFLD) and CF-related diabetes (CFRD) were not statistically different. The levels of glycemia and C- reactive protein were not statistically different. None of these pa- tients underwent transplantation. Furthermore, no patient had al- terations of blood clotting parameters (i.e., PT and PTT).
Table 2 shows the baseline values of cholesterol metabolism and hepatobiliary injury/function evaluations in the homo- deltaF508 patients in comparison to the hetero-deltaF508. Baseline
Patient characteristics from homo-deltaF508 (n = 20) and hetero-deltaF508 (n = 20).
Parameters Homo-deltaF508 Hetero-deltaF508
Age (years) 27 (7) 28 (6)
Male, n (%) 10 (50) 9 (45)
BMI (kg/m2 ) 21.3 (20.1–22.5) 21.0 (20.2–22.3)
ence limit. Vitamin E levels were significantly lower in the homo- deltaF508 group.
Then we evaluated the effect of lumacaftor/ivacaftor treatment on cholesterol metabolism and hepatobiliary injury/function in homo-deltaF508 patients (Table 3). After the treatment, plasma cholesterol (99 vs 112 mg/dL; p < 0.05) and lathosterol (0.23 vs. 0.40 mg/dL; p < 0.0001) levels were significantly lower than
- deltaF508/4016inst PI, n (%)
20 (100) –
8 (40) 4 (20) 2 (10) 2 (10) 1 (5)
1 (5) 1 (5)
baseline levels. There was no difference in phytosterol concen- trations after lumacaftor/ivacaftor treatment. On the other hand, the cholestanol concentrations were significantly higher after the lumacaftor/ivacaftor treatment (0.32 vs 0.24 mg/dL; p < 0.05). Cholestanol levels were negatively correlated with lathosterol lev- els in homo-deltaF508, before as well as after the treatment (Sup-
plementary material, Figure S1, n = 40; p = 0.006). Vitamin E sig- nificantly increased (+25%) in patients after lumacaftor/ivacaftor treatment (p < 0.001), reaching a mean value comparable to that
PERT (units of lipase/g of fat) 3212 (1456)
Vitamin E suppl. (mg/day) a 362 (0)
CFLD, n (%) 4 (20)
CFRD, n (%) 9 (45)
2519 (1631) 353 (29)
2 (10) 10 (50)
observed in hetero-deltaF508 patients (Table 2). There was a corre- lation between the differences of vitamin E levels after and before lumacaftor/ivacaftor treatment (ti Vitamin E) and the months of
Glycemia (mg/dL) b hs-CRP (mg/L) c
90 (79–101) 93 (77–99)
0.49 (0.33–0.90) 0.42 (0.33–1.55)
drug administration (Fig. 1; r = 0.569, n = 20, p = 0.009).
Lumacaftor/ivacaftor treatment caused a reduction (about
Continuous normal data are reported as mean (SD) and statistical dif- ferences were evaluated by unpaired Student t-test. Continuous non- parametric data are reported as median (interquartile range) and statistical differences were evaluated by Mann-Whitney U test. Categorical data are re- ported as frequency (percentage) and chi-square test was used to compare the frequencies.
CFLD: CF-associated liver disease; CFRD: CF-related diabetes; homo- deltaF508: CF patients homozygous for deltaF508del mutation; hetero- deltaF508: CF patients compound heterozygous for deltaF508del mutation and another severe mutation; hs-CRP: high sensitivity C-reactive protein; PI: pancreatic insufficiency; PERT: pancreatic enzyme replacement therapy.
a Supplementation of vitamin E by multivitamin complex.
b Reference range: 70–110 mg/dL.
c Reference range: 0–0.5 mg/L.
cholesterol plasma levels were significantly lower in the homo- deltaF508 group compared to the hetero-deltaF508 group (112 vs. 132 mg/dL, respectively; p < 0.05) and below the lower refer-
-38%) of serum AP. In particular, two patients had AP of 278 U/L (affected by cirrhosis) and 384 U/L (affected by CFLD) before the treatment, and, after 21 and 9 months of treatment, AP values re- duced at 86 U/L (-69%) and 255 U/L (-34%) U/L, respectively.
- Discussion and conclusions
In this study we evaluated the effects of lumacaftor/ivacaftor on cholesterol metabolism and hepatobiliary injury/function in homo-deltaF508 patients. Baseline plasma cholesterol and vitamin E levels were significantly lower in these patients than in hetero- deltaF508 patients, although patients in both groups were taking the same dose of PERT and vitamin E. These results could be due to the need of patients with homo-deltaF508 to take a higher PERT dose to obtain a greater availability of absorbable cholesterol. It is well known that pancreatic function is worse and more variable in homo-deltaF508 than in hetero-deltaF508 patients .
Cholesterol metabolism and hepatobiliary injury/function at basal state in homo-deltaF508 (n = 20) vs. hetero-deltaF508 patients (n = 20).
homo-deltaF508 hetero-deltaF508 Reference intervals Lipids (mg/dL)
Cholesterol 112 (26)∗ 132 (42) 121–232
HDL-cholesterol 43 (37–53) 49 (34–55) > 40
LDL-cholesterol 58 (47–72) 58 (52–74) < 115
Triglycerides 62 (54–73) 74 (63–95) < 150
Phytosterols 0.27 (0.13) 0.31 (0.19) 0.26–1.22
Lathosterol 0.40 (0.14) 0.46 (0.20) 0.12–0.49
Cholestanol 0.24 (0.12) 0.23 (0.13) 0.09–0.45
Vitamin E (μg/dL) 886 (215)∗ 1167 (450) 500–1800
AST (U/L) 20 (18–24) 19 (16–29) 0–34
ALT (U/L) 21 (17–25) 22 (18–30) 0–55
γ GT (U/L) 14 (10–18) 14 (11–23) 12–64
AP (U/L) 110 (98–118) 122 (105–174) 40–150
Total bilirubin (mg/dL) 0.60 (0.40–0.82) 0.50 (0.40–0.79) 0.20–1.20
Direct bilirubin (mg/dL) 0.29 (0.22–0.36) 0.26 (0.20–0.36) 0–0.40
Albumin (g/dL) 4.3 (0.3) 4.2 (0.4) 3.5–5.2
Continuous normal data are reported as mean (SD) and statistical differences were evalu- ated by unpaired Student t-test. Continuous non-parametric data are reported as median (interquartile range) and statistical differences were evaluated by Mann-Whitney U test. ∗ p
< 0.05, homo-deltaF508 vs. hetero-deltaF508.
ALT, alanine transaminase; AP: alkaline phosphatase; AST, aspartate aminotransferase; γ GT, γ -glutamyltransferase; homo-deltaF508: CF patients homozygous for deltaF508del mutation; hetero-deltaF508: CF patients compound heterozygous for deltaF508del mutation and an- other severe mutation.
Effects of lumacaftor/ivacaftor on cholesterol metabolism and hepatobiliary injury/function in homo-deltaF508 patients (n = 20).
Before After Reference intervals Lipids (mg/dL)
Cholesterol 112 (26) 99 (14)∗ 121–232
HDL-cholesterol 43 (37–53) 50 (42–58) > 40
LDL-cholesterol 58 (47–72) 53 (49–60) < 115
Triglycerides 62 (54–73) 64 (54–69) < 150
Phytosterols 0.27 (0.13) 0.24 (0.06) 0.26–1.22
Lathosterol 0.40 (0.14) 0.23 (0.07)∗∗∗ 0.12–0.49
Cholestanol 0.24 (0.12) 0.32 (0.07)∗ 0.09–0.45
Vitamin E (μg/dL) 886 (215) 1112 (309)∗∗ 500–1800
AST (U/L) 20 (18–24) 22 (19–27) 0–34
ALT (U/L) 21 (17–25) 18 (14–26) 0–55
γ GT (U/L) 14 (10–18) 12 (10–15) 12–64
AP (U/L) 110 (98–118) 68 (60–84)∗∗ 40–150
Total bilirubin (mg/dL) 0.60 (0.40–0.82) 0.36 (0.33–0.53) 0.20–1.20
Direct bilirubin (mg/dL) 0.29 (0.22–0.36) 0.18 (0.16–0.21) 0–0.40
Albumin (g/dL) 4.3 (0.3) 4.6 (0.4) 3.5–5.2
Continuous normal data are reported as mean (SD) and statistical differences were evaluated by paired Student t-test. Continuous non-parametric data are reported as median (interquar- tile range) and statistical differences were evaluated by paired Wilcoxon test. ∗ p < 0.01, ∗∗ p
< 0.005, ∗∗∗ p < 0.0001, after vs before treatment.
ALT, alanine transaminase; AP: alkaline phosphatase; AST, aspartate aminotransferase; γ GT, γ -glutamyltransferase.
Fig. 1. Correlation of the difference between serum vitamin E levels after and before lumacaftor/ivacaftor treatment (ti Vitamin E) vs. the months of treatment (n = 20; r = 0.569, p = 0.009).
After lumacaftor/ivacaftor treatment, cholesterol plasma con- centrations further decreased (-11.6%), even if we should also take
into account that the total variability (biological and analytical) of this parameter is about 10–12% [23,24]. The treatment signifi- cantly reduces also the levels of plasma lathosterol that became comparable with those obtained in healthy subjects [1,2], indi- cating a normalization of liver cholesterol biosynthesis in treated homo-deltaF508 patients [1,2]. These effects are relevant, because in patients with CF there is an impairment of the mechanism of cholesterol secretion by the liver in blood probably due to the dys- function of the CFTR protein [4,5]. So, the enhanced endogenous biosynthesis may cause the accumulation of cholesterol due to a potential altered trafficking in liver cells triggering inflammation, as observed in CF epithelial cell models . However, it is unclear why cholesterol synthesis is elevated in CF liver, as various studies
demonstrate that CFTR is not expressed in hepatocytes, but mainly in the bile duct epithelial cells. Therefore, a functional cross-talk between cholangiocytes and hepatocytes or a probable CFTR ex- pression in hepatocytes at low-level could be speculated . The enhanced liver synthesis of cholesterol could be induced by the re- duced influx from the intestine as well as availability of cholesterol for bile acid synthesis but other hypotheses are also plausible such as a reduced and/or an impaired trafficking of cholesterol in cell compartments.
After the treatment with lumacaftor/ivacaftor, the levels of plasma cholestanol, a surrogate marker of intestinal absorption , were significantly increased, suggesting that the drug im- proves the mechanism of intestinal cholesterol absorption in pa- tients with CF , although phytosterols levels did not increase after the treatment. Jakulj et al.  questioned the role of phy-
tosterols as marker of intestinal cholesterol absorption. However, it is suggested to evaluate simultaneously multiple markers of ab- sorption and synthesis, as in some clinical conditions absolute or relative markers may lose validity , and this could represent a limitation of this study.
Interestingly, we observed a significant increase of vitamin E levels after the treatment and the ti Vitamin E was positively cor- related with the months of treatment. These findings may sug- gest a significant effect of drug on intestinal digestion/absorption of lipids .
Overall these results suggest that the plasma cholesterol reduc- tion in lumacaftor/ivacaftor-treated homo-deltaF508 patients may depend in part on the reduction of endogenous biosynthesis, but also on a probable increase of bile salt production and/or excre- tion. Sterols and vitamin E are strongly dependent on bile salt mi- cellization for intestinal absorption , and the increase of vi- tamin E and cholestanol after lumacaftor/ivacaftor treatment may suggest an adequate presence of bile salts in intestinal lumen. Therefore, the decreased hepatic cholesterol synthesis and a prob- able increased bile salts production could influence more liver metabolism of cholesterol than intestinal absorption of sterols re- sulting in a reduction of blood cholesterol.
Our study shows that the treatment with lumacaftor/ivacaftor significantly reduces the levels of AP suggesting a positive effect of the drug on the hepato-canalicular inflammation [25,29]. In par- ticular, this effect has been greatly improved in 2 patients with a form of CFLD . It is also necessary to remember that AP is not only a marker of cholangiocytic damage, but its serum lev- els also vary in all those physiological and pathological condi- tions involving the bone tissue, including changes induced by bone metabolism regulatory molecules, such as vitamin D, PTH and cal- citonin. Our data contrast with a previous study that reported al-
Declaration of competing interest
None of the authors have any relevant conflicts of interest to disclose.
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jcf.2020.06.015.
CRediT authorship contribution statement
Monica Gelzo: Data curation, Methodology, Visualization, Writ- ing - original draft. Paola Iacotucci: Investigation, Data curation. Mafalda Caputo: Methodology. Gustavo Cernera: Formal analysis. Marika Comegna: Formal analysis. Vincenzo Carnovale: Investi- gation. Gaetano Corso: Conceptualization, Supervision, Validation, Writing - review & editing. Giuseppe Castaldo: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Visualization, Writing - review & editing.
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Overall, these data suggest an effect of lumacaftor/ivacaftor on enterohepatic flux and on cholesterol metabolism in patients with CF. However, these effects are unable to correct the low levels of plasma cholesterol observed in most patients with CF  that could expose such patients to a series of severe consequences [31,32].
The effect of lumacaftor/ivacaftor observed in this study may suggests that the drug, acting on the conformation of the protein CFTR, induces an improvement in the flows of intra-extra-cellular sterols, through mechanisms not yet known, but with positive ef- fects both on intestinal cells (improvement of absorption) and on hepatocytes (reduction of synthesis and/or increase of sterols ex- cretion).
In conclusion, our preliminary study shows that lumacaftor/ivacaftor changes cholesterol metabolism in patients with CF. In particular, the analysis of lathosterol, cholestanol and vitamin E allows to evaluate the effects of lumacaftor/ivacaftor on hepatic cholesterol synthesis and intestinal lipid absorption. How- ever, lumacaftor/ivacaftor is unable to increase cholesterolemia that on the contrary declines. Further studies are needed to investigate the real mechanism causing this reduction.
This work was supported by Ministero della Salute (Rome, Italy)
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