Differential distribution of ivacaftor and its metabolites in plasma and human airway epithelia.

Ivacaftor is the first clinically approved monotherapy potentiator to treat CFTR channel dysfunction in people with cystic fibrosis. Ivacaftor (Iva) is a critical component for all current modulator therapies, including highly effective modulator therapies. Clinical studies show that CF patients on ivacaftor-containing therapies present various clinical responses, off-target effects, and adverse reactions, which could be related to metabolites of the compound. In this study, we reported the concentrations of Iva and two of its major metabolites (M1-Iva and M6-Iva) in capillary plasma and estimated M1-Iva and M6-Iva metabolic activity via the metabolite parent ratio in capillary plasma over 12 hours. We also used the ratio of capillary plasma versus human nasal epithelial cell concentrations to evaluate entry into epithelial cells in vivo. M6-Iva was rarely detected by LC-MS/MS in epithelial cells from participants taking ivacaftor, although it was detected in plasma. To further explore this discrepancy, we performed in vitro studies, which showed that M1-Iva, but not M6-Iva, readily crossed 16HBE cell membranes. Our studies also suggest that metabolism of these compounds is unlikely to occur in airway epithelia despite evidence of expression of metabolism enzymes. Overall, our data provide evidence that there are differences between capillary and cellular concentrations of these compounds that may inform future studies of clinical response and off-target effects.

Pharmacokinetic variability of CFTR modulators from standard and alternative regimens.

Elexacaftor, tezacaftor, ivacaftor (ETI) is a CFTR modulator combination approved for use in ∼90 % of people with cystic fibrosis (pwCF) over 2 years old. While most pwCF tolerate this therapy well, some are intolerant to standard dosing, and others show little response. Clinical providers may adjust ETI dosing to combat these issues, but these adjustments are not well guided by pharmacokinetic evidence. Our post-approval study aimed to describe pharmacokinetic variability of ETI plasma concentrations in 15 participants who were administered a standard or reduced dose. ETI were quantified by LC-MS/MS in plasma samples taken prior to the morning dose. Results showed non-significant differences for each compound regardless of dosing regimen and after dose equivalence normalization. The majority of participants in both dosing groups had concentrations expected to elicit clinical response to ETI therapy. These findings indicate that dose reduction may be a viable strategy to maintain clinical benefit while managing intolerance.

CFTR function and clinical response to modulators parallel nasal epithelial organoid swelling.

In vitro biomarkers to assess cystic fibrosis transmembrane conductance regulator activity are desirable for precision modulator selection and as a tool for clinical trials. Here, we describe an organoid swelling assay derived from human nasal epithelia using commercially available reagents and equipment and an automated imaging process. Cells were collected in nasal brush biopsies, expanded in vitro, and cultured as spherical organoids or as monolayers. Organoids were used in a functional swelling assay with automated measurements and analysis, whereas monolayers were used for short-circuit current measurements to assess ion channel activity. Clinical data were collected from patients on modulators. Relationships between swelling data and short-circuit current, as well as between swelling data and clinical outcome measures, were assessed. The organoid assay measurements correlated with short-circuit current measurements for ion channel activity. The functional organoid assay distinguished individual responses as well as differences between groups. The organoid assay distinguished incremental drug responses to modulator monotherapy with ivacaftor and combination therapy with ivacaftor, tezacaftor, and elexacaftor. The swelling activity paralleled the clinical response. In conclusion, an in vitro biomarker derived from patients' cells can be used to predict responses to drugs and is likely to be useful as a preclinical tool to aid in the development of novel treatments and as a clinical trial outcome measure for a variety of applications, including gene therapy or editing.

Electronic screening for unmet social needs in a pediatric pulmonary clinic: Acceptability and associations with health outcomes.

BACKGROUND: Children with unmet basic needs experience worse health than more advantaged counterparts. There has been limited research on screening for unmet basic needs in pediatric subspecialty care. METHODS: Caregivers of established patients in pediatric asthma and cystic fibrosis (CF) clinics were screened for unmet basic needs with an electronic survey, which asked about concerns and stress level (5-point Likert scale) related to food, housing, transportation, health insurance, and childcare, among others. Medical record review provided patient demographic characteristics and clinical data. A follow-up survey with the clinical providers assessed the acceptability of electronic screening for unmet needs. RESULTS: The sample included 214 pediatric patients (N = 105 asthma, N = 109 CF) and their caregivers. Most patients with asthma (76%) were Black, 30% in households with <$20,000 annual income. In contrast, most patients with CF (93%) were white, 12% in households with <$20,000 annual income. Reported needs included food insecurity (29% asthma and 17% CF), healthy food (75% asthma and 87% CF), financial insecurity (45% asthma and 32% CF), health insurance (15% asthma and 28% CF), smoke exposure (24% asthma and 28% CF), child's exercise (21% asthma and 28% CF), living conditions (18% asthma and 17% CF), childcare (11% asthma and 15% CF), transportation (16% asthma and 9% CF), and housing insecurity (10% asthma and 8% CF). Concerns were rated moderately to very stressful. Food insecurity, financial insecurity, and smoke exposure were significantly associated with uncontrolled asthma. In people with CF, concerns about health insurance and child exercise were significantly associated with lower lung function and increased odds of hospitalizations. Clinicians believed that screening was important and should be administered by a designated person on the clinical team. CONCLUSIONS: Unmet basic needs and associated stress levels are linked to adverse pediatric pulmonary outcomes. Electronic screening, without face-to-face interaction or paper trail, facilitates high response rates and is easily integrated into clinic flow. Such screenings can identify vulnerable patients for targeted interventions and referral to available community resources.

Outcomes of children with cystic fibrosis screen positive, inconclusive diagnosis/CFTR related metabolic syndrome.

Background: Some infants undergoing newborn screening (NBS) tests have inconclusive sweat chloride test (SCT) results that lead to the designation of Cystic Fibrosis Screen Positive, Inconclusive Diagnosis/CFTR-related metabolic syndrome (CFSPID/CRMS). Some proportion of them transition to a CF diagnosis, but no predictive markers can stratify which are at risk for this transition. We report single-center outcomes of children with CRMS. Methods: We retrospectively identified all infants born in Alabama from 2008 through 2020 referred to our CF Center with an elevated immunoreactive trypsinogen level (IRT) associated with a cystic fibrosis transmembrane conductance regulator (CFTR) mutation (IRT+/DNA+) who had at least one SCT result documented. Infants were classified per established guidelines as Carrier, CRMS, or CF based on the IRT+/DNA+ and SCT results. The electronic health record was reviewed for follow-up visits until the children received a definitive diagnosis (to carrier or CF) according to current diagnostic guidelines for CF, or through the end of the 2020 year. Results: Of the 1,346 infants with IRT+ and at least 1 CFTR mutation identified (IRT+/DNA+), 63 (4.7%) were designated as CRMS. Of these infants, 12 (19.1%) transitioned to Carrier status (CRMS-Carrier), 40 (63.5%) of them remained CRMS status (CRMS-Persistent) and 11 (17.5%) of them transitioned to a diagnosis of CF (CRMS-CF). Of the 11 children in the CRMS-CF group, 4 (36%) had an initial SCT 30-39 mmol/L, 4 (36%) had an initial SCT 40-49 mmol/L and 3 (27%) had an initial SCT 50-59 mmol/L. These children also had higher initial sweat tests and greater yearly increases in sweat chloride values than others with CRMS. We found that in comparison to children in the CRMS-P group, a greater proportion of children in the CRMS-CF group cultured bacteria like methicillin-resistant Staphylococcus aureus, Stenotrophomonas maltophilia, and Pseudomonas aeruginosa, had smaller weight-for-height percentiles and remained smaller over time despite slightly greater growth. Conclusion: Infants with an inconclusive diagnosis of CF should continue to receive annual care and management given their potential risk of transition to CF. Further research is needed to assess whether certain phenotypic patterns, clinical symptoms, diagnostic tests or biomarkers could better stratify these children.

Mobile health platform for self-management of pediatric cystic fibrosis: Impact on patient-centered care outcomes.

BACKGROUND: Previously, we adapted a mobile health platform (Genia) to the needs of patients and families in a pediatric CF center in the United States. In this feasibility study, we tested the impact of Genia on measures of patient-centered care. METHODS: In a one-group pre-post study with adolescents with CF and caregivers of children with CF, we tested Genia's effect over 6 months on patient satisfaction with chronic illness care (PACIC) and shared decision-making (CollaboRate). Feasibility and acceptability were assessed with exit interviews and app analytics. RESULTS: The intervention included 40 participants: 30 caregivers of children with CF age ≤14 years and 10 patients with CF age ≥15 years. The use of Genia was associated with increased satisfaction with care (p = 0.024), including delivery system and decision support (p = 0.017), goal setting (p = 0.006), and shared decision-making (p<0.001). The use of Genia was associated with nominal improvements in all QOL domains and symptom scales. The platform was feasible, with participants recording more than 4,400 observations (mean 84.2) and submitting 496 weekly reports (mean 13.8) and 70 quarterly reports (mean 1.8), and acceptable (95% retention rate). For participants, the most useful app feature was pre-visit reports (66.7%), and the top symptom trackers were those for cough (23.7%), appetite (21.1%), energy (18.4%), and medicines (18.4%). CONCLUSION: The use of Genia over 6 months was feasible, acceptable, and associated with improved measures of patient-centered care. Study results support wider use of Genia in clinical settings. Efficacy for clinical outcomes should be assessed in a randomized clinical trial.

Pharmacogenetic actionability and medication prescribing in people with cystic fibrosis.

Although major advancements have been made in the therapeutics for people with cystic fibrosis (PwCF), many still require the use of multiple medications to manage acute exacerbations of disease and maintain health. Iterative trial and error processes of pharmacotherapeutic management can be optimized by assessing and incorporating pharmacogenetics. For 82 PwCF, we reviewed 2 years of medication use and response history and interrogated metabolizer status for common pharmacogenes, revealing 3336 medication exposure events (MEEs) to 286 unique medications. As expected, the more frequent MEEs were those commonly used to treat cystic fibrosis (CF), such as antibiotics and respiratory medications. Antibiotics also comprised 56.7% of the undesirable drug responses. The impact of gene variants on drug responses was assessed using Pharmacogenomics Knowledgebase (PharmGKB) and Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines. Thirty-three (11.5%) medications have strong evidence of genetic influence on response as evidenced by gene-based dosing guidelines. 110 (38.5%) unique medications had at least one association with a very important pharmacogene, whereas 143 (50%) were associated with at least one clinical or variant annotation. Over 97% of participants had at least one actionable genotype. Eleven (13.4%) patients with an actionable genotype, taking the impacted medication, had an undesirable drug response described in the CPIC guidelines that could potentially have been mitigated with a priori knowledge of the genotype. PwCF take many medications for disease management, with frequent dose changes to elicit a desired clinical effect. As CF care evolves, implementing pharmacogenetics testing can improve efficiency and safety of prescribing practices using precision selection and dosing at medication initiation.

Novel Applications of Biomarkers and Personalized Medicine in Cystic Fibrosis.

As routine care in cystic fibrosis (CF) becomes increasingly personalized, new opportunities to further focus care on the individual have emerged. These opportunities are increasingly filled through research in tools aiding drug selection, drug monitoring and titration, disease-relevant biomarkers, and evaluation of therapeutic benefits. Herein, we will discuss such research tools presently being translated into the clinic to improve the personalization of care in CF.

Ivacaftor-elexacaftor-tezacaftor and tacrolimus combination in cystic fibrosis.

The CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ETI) is a genetic mutation-targeted treatment in cystic fibrosis that results in profound improvements in clinical outcomes. Each of the compounds are substrates of CYP3A4/5, the cytochrome P450 enzyme family for which tacrolimus is also a substrate. The use of these compounds in an individual with a solid organ transplant has not been previously studied and there is potential for a drug interaction. In this report, we describe a pediatric liver transplant recipient with clinical decline related to cystic fibrosis who improved substantially with ETI, without significant impact on the systemic exposure of either ETI or tacrolimus.

Plasma and cellular ivacaftor concentrations in patients with cystic fibrosis.

Access to cystic fibrosis transmembrane conductance regulator (CFTR) modulators has been gradually increasing for people with cystic fibrosis, the first of which was ivacaftor, a CFTR potentiator that is part of all clinically available modulator treatments. In this study, we hypothesized that the steady-state concentrations in blood and tissue are highly variable in patients taking ivacaftor in a real-world context, which may have an impact on the treatment approach. We collected nasal epithelial cells to estimate target site concentrations and blood samples to estimate pharmacokinetic parameters at a steady state. We found that patients on ivacaftor monotherapy have variable concentrations well above the maximal effective concentration and may maintain concentrations necessary for the clinical benefit even if dosing is reduced. We also are the first to provide detailed target site concentration data over time, which shows that tissue concentrations do not fluctuate significantly and do not correlate with plasma concentrations. These findings show that some patients may have higher-than-expected concentrations and may benefit from tailored dosing to balance clinical response with side effects or adherence needs.

Use of elexacaftor/tezacaftor/ivacaftor among cystic fibrosis lung transplant recipients.

BACKGROUND: Cystic fibrosis (CF) lung transplant (LT) recipients may warrant treatment with elexacaftor/tezacaftor/ivacaftor (ETI) to improve extrapulmonary manifestations of CF. Our objectives were to identify reasons for prescribing ETI after LT and evaluate changes in body mass index (BMI), hemoglobin A1c, hemoglobin, and liver enzymes. METHODS: This was an electronic health record-based cohort study, October 2019-September 2020, at 14 CF LT Consortium sites in North America. The study included CF LT recipients prescribed ETI after transplant. Differences in BMI, A1c, and hemoglobin were assessed with paired t-tests. RESULTS: There were 94 patients prescribed ETI; indications included sinus disease (68%), GI symptoms (39%), or low BMI (19%). Prescriptions were written by CF physicians (34%), LT physicians (27%), or physicians who practice both CF and LT (39%). Forty patients (42%) stopped ETI at a median of 56 days [IQR 26, 139] after start/prescription date. ETI was not associated with a significant change in BMI (0.2 kg/m2, 95% CI [-0.1, 0.6], p = 0.150), but was associated with decreased A1c (0.4%, 95% CI 0.2, 0.7, p = 0.003), and increased hemoglobin for patients with anemia (0.6 g/dL, 95% CI 0.2, 1.0, p = 0.007). Three people (3%) stopped ETI due to elevated transaminases. CONCLUSIONS: ETI is rarely prescribed for non-pulmonary indications after LT for CF. Further study is needed to determine the risks and benefits of ETI in the CF lung transplant population given the potential for drug interactions, side effects leading to discontinuation of ETI, and the possible mechanisms for ETI to positively impact long-term post-transplant outcomes.

Quantitation of cystic fibrosis triple combination therapy, elexacaftor/tezacaftor/ivacaftor, in human plasma and cellular lysate.

The triple combination modulator therapy (ETI, elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA)) is a recent advancement for the care of patients with cystic fibrosis. To aid in the development of clinical pharmacokinetics studies of this treatment, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantifying the component compounds in human plasma and cell lysate. This assay was optimized for small volumes (10 µL), uses stably labeled isotopes of the ETI compounds as internal standards, and employs a simple methanol protein precipitation method. Chromatography was performed on an ACE Excel C18, 2.1 × 50 mm, reversed phase analytical column, using a step or bump isocratic method, with mobile phases consisting of 0.1% formic acid in water for A, and 0.1% formic acid in acetonitrile for B. Analyte and internal standard detection was conducted with ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 598.0/422.0 for ELX, 521.0/449.0 for TEZ, 393.0/172.0 for IVA, 601.0/422.0 for IS-ELX, 525.0/453.0 for IS-TEZ, and 399.0/178.0 for IS-IVA, respectively. The assay has a dynamic range of 10 to 10,000 ng/mL, with a mean coefficient of determination (r2, mean ± SD) of 0.9970 ± 0.0027 (ELX), 0.9989 ± 0.0004 (TEZ), 0.9981 ± 0.0003 (IVA), regardless of specimen matrix. The mean precision values for all calibration standards ranged from 0.0 to 10.8% (ELX), 0.0 to 6.7% (TEZ), and 0.2 to 5.6% (IVA), while the accuracy for calibration standards was within the range of -5.7 to 3.5% (ELX), -3.2 to 6.0% (TEZ), and -3.8 to 5.2% (IVA). Validation results demonstrated high accuracy (≤7.3, ≤9.8, ≤10.6% deviation) and high precision (≤11.5, ≤6.3, ≤11.0% CV) for the respective ETI quality control samples. This method provides a fully validated assay for ETI quantitation for use in clinical research.

Culture and Imaging of Human Nasal Epithelial Organoids.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

Human Nasal Epithelial Organoids for Therapeutic Development in Cystic Fibrosis.

We describe a human nasal epithelial (HNE) organoid model derived directly from patient samples that is well-differentiated and recapitulates the airway epithelium, including the expression of cilia, mucins, tight junctions, the cystic fibrosis transmembrane conductance regulator (CFTR), and ionocytes. This model requires few cells compared to airway epithelial monolayer cultures, with multiple outcome measurements depending on the application. A novel feature of the model is the predictive capacity of lumen formation, a marker of baseline CFTR function that correlates with short-circuit current activation of CFTR in monolayers and discriminates the cystic fibrosis (CF) phenotype from non-CF. Our HNE organoid model is amenable to automated measurements of forskolin-induced swelling (FIS), which distinguishes levels of CFTR activity. While the apical side is not easily accessible, RNA- and DNA-based therapies intended for systemic administration could be evaluated in vitro, or it could be used as an ex vivo biomarker of successful repair of a mutant gene. In conclusion, this highly differentiated airway epithelial model could serve as a surrogate biomarker to assess correction of the mutant gene in CF or other diseases, recapitulating the phenotypic and genotypic diversity of the population.

Variable cellular ivacaftor concentrations in people with cystic fibrosis on modulator therapy.

The development of CFTR modulators has transformed the care of patients with cystic fibrosis (CF). Although the clinical efficacy of modulators depends on their concentrations in target tissues, the pharmacokinetic properties of these drugs in epithelia are not utilized to guide patient care. We developed assays to quantitate ivacaftor in cells and plasma from patients on modulator therapy, and our analyses revealed that cellular ivacaftor concentrations differ from plasma concentrations measured concurrently, with evidence of in vivo accumulation of ivacaftor in the cells of patients. While the nature of this study is exploratory and limited by a small number of patients, these findings suggest that techniques to measure modulator concentrations in vivo will be essential to interpreting their clinical impact, particularly given the evidence that ivacaftor concentrations influence the activity and stability of restored CFTR protein.

Co-cultured microfluidic model of the airway optimized for microscopy and micro-optical coherence tomography imaging.

We have developed a human bronchial epithelial (HBE) cell and endothelial cell co-cultured microfluidic model to mimic the in vivo human airway. This airway-on-a-chip was designed with a central epithelial channel and two flanking endothelial channels, with a three-dimensional monolayers of cells growing along the four walls of the channel, forming central clear lumens. These cultures mimic airways and microvasculature in vivo. The central channel cells are grown at air-liquid interface and show features of airway differentiation including tight-junction formation, mucus production, and ciliated cells. Combined with novel micro-optical coherence tomography, this chip enables functional imaging of the interior of the lumen, which includes quantitation of cilia motion including beat frequency and mucociliary transport. This airway-on-a chip is a significant step forward in the development of microfluidics models for functional imaging.

Role of oxygen availability in CFTR expression and function.

The cystic fibrosis transmembrane conductance regulator (CFTR) serves a pivotal role in normal epithelial homeostasis; its absence leads to destruction of exocrine tissues, including those of the gastrointestinal tract and lung. Acute regulation of CFTR protein in response to environmental stimuli occurs at several levels (e.g., ion channel phosphorylation, ATP hydrolysis, apical membrane recycling). However, less information is available concerning the regulatory pathways that control levels of CFTR mRNA. In the present study, we investigated regulation of CFTR mRNA during oxygen restriction, examined effects of hypoxic signaling on chloride transport across cell monolayers, and related these findings to a possible role in the pathogenesis of chronic hypoxic lung disease. CFTR mRNA, protein, and function were robustly and reversibly altered in human cells in relation to hypoxia. In mice subjected to low oxygen in vivo, CFTR mRNA expression in airways, gastrointestinal tissues, and liver was repressed. CFTR mRNA expression was also diminished in pulmonary tissues taken from hypoxemic subjects at the time of lung transplantation. Environmental factors that induce hypoxic signaling regulate CFTR mRNA and epithelial Cl(-) transport in vitro and in vivo.

Sensitivity of ivacaftor to drug-drug interactions with rifampin, a cytochrome P450 3A4 inducer.

The CFTR potentiator ivacaftor is responsible for significant clinical improvements among a subset of patients with cystic fibrosis. Because it is a substrate of the cytochrome P450 system, specifically CYP3A4/5, ivacaftor is subject to significant drug-drug interactions, including due to commonly used antimicrobials such as rifampin. While the interaction of rifampin and ivacaftor has been examined in vitro, severe adverse events resulting from this interaction have not been reported in the literature. In this report, we describe the termination of steady, long-term improvement in a patient taking ivacaftor, resulting from the use of rifampin and precipitating a significant pulmonary exacerbation.

Potential pathogenicity of Inquilinus limosus in a pediatric patient with cystic fibrosis.

PRESENTATION: Patient is a 6-year-old male with CF, MRSA colonization, and pancreatic insufficiency that presented with worsening ppFEV1 and systemic symptoms despite multiple interventions. BAL grew NTM, Stenotrophomonas maltophilia, and Inquilinus limosus, a rare organism found in patients with CF. COURSE: I. limosus treatment was deferred. Despite treatment of other pathogens, symptoms worsened. I. limosus was targeted with meropenem, amikacin, and ciprofloxacin along with clindamycin for MRSA colonization. Within weeks, symptoms had resolved with ppFEV1 improvement. DISCUSSION: This case discusses the importance of a rare organism in the CF population. Targeting I. limosus was key to recovery, revealing its potential pathogenicity.