An evaluation of the pharmacokinetics of tiotropium following a single-dose inhalation in healthy subjects using an ultra-sensitive bioanalytical method.

Objective and significance: The systemic bioavailability of tiotropium following administration via inhalation is known to be very low. A validated ultra-sensitive bioanalytical method with the lowest lower limit of quantitation (LLOQ) was developed and used to evaluate the complete pharmacokinetic profile of tiotropium.Methods: This was a pharmacokinetic study performed in 18 healthy subjects. Each subject was administered a dose of 18 mcg of tiotropium from a dry powder inhaler (DPI). The subjects' plasma tiotropium concentrations were assayed with LLOQ of 0.1 pg/mL.Results: The results showed a mean Cmax of 4.98 ± 3.55 pg/mL, and a median (tmax) of 3.6 minutes (range: 1.8-12 minutes). The means for area under the concentration-time curve (AUC) from time zero hours to infinity (AUCinf) and AUC from time zero hours to the time of the last measurable tiotropium concentration (AUCt) were 51.11 ± 27.4 pg*h/mL and 37.37 ± 23.38 pg*h/mL, respectively. The mean apparent elimination half-life (t1/2) was 68.02 ± 24.55 hours. This calculated half-life is longer than what others have reported where a less sensitive LLOQ was used.Conclusion: The lower LLOQ enabled further insight into the pharmacokinetics of tiotropium that was not possible with other analytical methods. With this method, we were able to quantify tiotropium concentrations as early as one minute following drug administration and up to 144 hours after dosing. The application of this method will allow for studies to be designed properly and enable further investigations into the pharmacokinetics of tiotropium.

Bronchodilator efficacy of tiotropium/formoterol (18/12 µg once daily via a Discair inhaler), tiotropium alone (18 µg by Handihaler) or combined with formoterol (12 µg twice daily by Aerolizer) in adults with moderate-to-severe stable COPD.

Objectives: The bronchodilator efficacy of a once-daily fixed-dose combination of tiotropium/formoterol (18/12 µg administered via a dry-powder inhaler, Discair) [TIO/FORMfixed group] vs a single-dose of tiotropium (18 µg) by Handihaler1 alone [TIOmono group], or combined with formoterol 12 µg twice-daily by Aerolizer2 [TIO/FORMbid group] was compared in patients with moderate-to-severe stable COPD.Methods: COPD patients were randomized (28 patients/group) to receive TIO/FORMfixed, TIOmono, or TIO/FORMbid. AUC for the changes in FEV1 and FVC over a 24-h period; bronchodilator response (100 ml improvement in FEV1) in the first 30 min; maximum changes in FEV1 and FVC; and safety data were recorded. The primary endpoint was to confirm the non-inferiority of TIO/FORMfixed vs TIO/FORMbid in terms of the AUC for the changes in FEV1 over a 24-h period.Results: Changes in AUC0-24h for FEV1 and FVC were similar for TIO/FORMfixed and TIO/FORMbid, and were superior to TIOmono (p < 0.001). A positive bronchodilator response at 30 min was demonstrated in 50%, 64%, and 71% of patients in the TIOmono, TIO/FORMbid, and TIO/FORMfixed groups, respectively (NS). Maximum FEV1 and FVC changes were measured as 0.25/0.41 L, 0.32/0.49 L, and 0.37/0.53 L, for TIOmono, TIO/FORMbid, and TIO/FORMfixed, respectively (FEV1: TIO/FORMfixed vs TIOmono, p = 0.0017 and TIO/FORMfixed vs TIO/FORMbid, p = 0.4846); no differences were recorded between the combination groups.Conclusions: The 24-h bronchodilator efficacy of TIO/FORMfixed 18/12 µg once-daily by Discair3 was non-inferior to a combination of tiotropium 18 µg by Handihaler plus formoterol 12 µg twice-daily by Aerolizer, and superior to tiotropium 18 µg monotherapy by Handihaler.

Particle size and gastrointestinal absorption influence tiotropium pharmacokinetics: a pilot bioequivalence study of PUR0200 and Spiriva HandiHaler.

AIMS: Plasma pharmacokinetics permit the assessment of efficacy and safety of inhaled drugs, and possibly their bioequivalence to other inhaled products. Correlating drug product attributes to lung deposited dose is important to achieving equivalence. PUR0200 is a spray-dried formulation of tiotropium that enables more efficient lung delivery than Spiriva® HandiHaler® (HH). The ratio of tiotropium lung-to-oral deposition in PUR0200 was varied to investigate the impact of particle size on tiotropium pharmacokinetics, and the contribution of oral absorption to tiotropium exposure was assessed using charcoal block. METHODS: A seven-period, single-dose, crossover study was performed in healthy subjects. PUR0200 formulations differing in dose and aerodynamic particle size were administered in five periods and Spiriva HH in two periods. In one period, Spiriva HH gastrointestinal absorption was blocked with oral charcoal. Tiotropium plasma concentrations were assessed over 8 h after inhalation. RESULTS: PUR0200 pharmacokinetics were influenced by aerodynamic particle size and the ratio of lung-to-oral deposition, with impactor sized mass (ISM) correlating most strongly with exposure. Formulation PUR0217a (3 µg tiotropium) lung deposition was similar to Spiriva HH (18 µg) with and without charcoal block, but total PUR0200 exposure was lower without charcoal. The Cmax and AUC0-0.5h of Spiriva HH with and without charcoal block were bioequivalent; however, Spiriva HH AUC0-8h was lower when gastrointestinal absorption was inhibited with oral charcoal administration. CONCLUSIONS: Pharmacokinetic bioequivalence indicative of lung deposition and efficacy can be achieved by matching the reference product ISM. Due to reduced oral deposition and more efficient lung delivery, PUR0200 results in a lower AUC0-t than Spiriva HH due to reduced absorption of drug from the gastrointestinal tract.

Risk of Severe Cardiovascular Events From Add-On Tiotropium in Chronic Obstructive Pulmonary Disease.

OBJECTIVE: To examine the risk of cardiovascular disease (CVD) from tiotropium added to inhaled long-acting ß2 agonists (LABAs) and inhaled corticosteroids (ICSs) in a nationwide population with chronic obstructive pulmonary disease (COPD). PATIENTS AND METHODS: This nested case-control study included 65,966 patients with COPD treated with LABAs and ICSs identified from the Taiwan nationwide health care claims database from January 1, 2007, through June 30, 2011. Cases were all patients with a first primary diagnosis of ischemic heart disease, heart failure, stroke, or arrhythmia from inpatient or emergency care settings during follow-up, and each was matched with 4 disease risk score-matched controls from risk sets. The use of tiotropium in the year before the index/event date was measured, stratified by duration since therapy initiation, concomitant COPD medications, and dosage form. Conditional logistic regression models were used to estimate odds ratios of the CVD risk from add-on tiotropium therapy. RESULTS: From the study cohort, with a mean age of 70.3 years (interquartile range, 61.8-79.4 years), 3188 CVD cases (incidence rate, 6.2 [95% CI, 6.0-6.4] cases per 100 person-years) and 12,349 matched controls were identified. The new use of tiotropium was associated with a 1.88-fold (95% CI, 1.44-2.46) increased CVD risk within 30 days of therapy initiation, and the association was sustained up to 60 days after treatment initiation (adjusted odds ratio, 1.71; 95% CI, 1.08-2.70). The risk persisted across all tiotropium regimens, with a case-crossover analysis, and in comparison with new add-on theophylline therapy. CONCLUSION: Tiotropium newly added to LABA/ICS combination therapy was associated with an increased cardiovascular risk in patients with COPD.

The pharmacokinetics, pharmacodynamics and tolerability of PUR0200, a novel tiotropium formulation, in chronic obstructive pulmonary disease.

AIMS: PUR0200 is a tiotropium bromide formulation engineered with the iSPERSE dry powder delivery technology. PUR0200 is being developed as a bioequivalent alternative to tiotropium bromide, delivered using Spiriva® HandiHaler® (HH). We investigated the bronchodilator effects, pharmacokinetics and safety of PUR0200 in patients with chronic obstructive pulmonary disease (COPD). METHODS: This was a randomized, placebo-controlled, crossover study using different PUR0200 doses and the comparator tiotropium HH. In vitro aerodynamic particle size distribution (aPSD) characterization of PUR0200 and tiotropium HH are presented. The main endpoints included forced expiratory volume in 1 s (FEV1 ) trough and (0-24 h) and pharmacokinetic parameters. RESULTS: The increased fine-particle fraction of PUR0200 demonstrated by testing using the next-generation impactor increased the proportion of drug available for lung deposition compared with the tiotropium HH. There was a numerical dose-response effect for PUR0200 on FEV1 , with 3 µg demonstrating a lower effect than higher doses. The placebo-adjusted mean (95% confidence interval) increases from baseline at 24 h postdose were 150 ml (100-200), 210 ml (160-270) and 200 ml (140-250) for 3 µg, 6 µg and 9 µg doses of PUR0200, respectively. Tiotropium HH (18 µg) caused a mean 169 ml (standard deviation 157ml) improvement in trough FEV1 , which was not significantly different to the PUR0200 effects at any of the tested doses. CONCLUSIONS: PUR0200 treatment caused bronchodilation in COPD patients that was similar in magnitude to that caused by tiotropium HH. This enabled a similar clinical effect on lung function to be achieved with PUR0200 using a lower metered dose of tiotropium compared with tiotropium HH.

In vitro and clinical characterization of the valved holding chamber AeroChamber Plus® Flow-Vu® for administrating tiotropium Respimat® in 1-5-year-old children with persistent asthmatic symptoms.

BACKGROUND: When characterizing inhalation products, a comprehensive assessment including in vitro, pharmacokinetic (PK), and clinical data is required. We conducted a characterization of tiotropium Respimat® when administered with AeroChamber Plus® Flow-Vu® anti-static valved holding chamber (test VHC) with face mask in 1-5-year-olds with persistent asthmatic symptoms. METHODS: In vitro tiotropium dose and particle size distribution delivered into a cascade impactor were evaluated under fixed paediatric and adult flow rates between actuation and samplings. The tiotropium mass likely to reach children's lungs was assessed by tidal breathing simulations and an ADAM-III Child Model. PK exposure to tiotropium in preschool children with persistent asthmatic symptoms (using test VHC) was compared with pooled data from nine Phase 2/3 trials in older children, adolescents, and adults with symptomatic persistent asthma not using test VHC. RESULTS: At fixed inspiratory flow rates, emitted mass and fine particle dose decreased under lower flow conditions; dose reduction was observed when Respimat® was administered by test VHC at paediatric flow rates. In <5-year-old children, such a dose reduction is appropriate. In terms of dose per kg/body weight, in vitro-delivered dosing in children was comparable with adults. Transmission and aerosol holding properties of Respimat® when administered with test VHC were fully sufficient for aerosol delivery to patients. At zero delay, particles <5 µm (most relevant fraction) exhibited a transfer efficacy of ≥60%. The half-time was>10 s, allowing multiple breaths. Standardized tidal inhalation resulted in an emitted mass from the test VHC of approximately one-third of labelled dose, independent of coordination and face mask use, indicating predictable tiotropium administration by test VHC with Respimat®. Tiotropium exposure in 1-5-year-old patients using the test VHC, when adjusted by height or body surface, was comparable with that in older age groups without VHCs; no overexposure was observed. Adverse events were less frequent with tiotropium (2.5 µg, n = 20 [55.6%]; 5 µg, n = 18 [58.1%]) than placebo (n = 25 [73.5%]). CONCLUSIONS: Our findings provide good initial evidence to suggest that tiotropium Respimat® may be administered with AeroChamber Plus® Flow-Vu® VHC in 1-5-year-old patients with persistent asthmatic symptoms. To confirm the clinical efficacy and safety in these patients, additional trials are required. CLINICAL TRIALS REGISTRY NUMBER: The trial was registered under NCT01634113 at http://www.clinicaltrials.gov.

Pharmacokinetics of tiotropium administered by Respimat® in asthma patients: Analysis of pooled data from Phase II and III clinical trials.

Tiotropium is a long-acting inhaled antimuscarinic bronchodilator that has recently received marketing authorization for the indication of asthma with dose delivery via the Respimat® inhaler, in addition to its widely established role in the management of chronic obstructive pulmonary disease (COPD). This report presents a combined analysis of tiotropium plasma and urine pharmacokinetics at steady state from 8 Phase II/III clinical trials in asthma and delineates the effects of patient characteristics on systemic exposure based on the parameters fe0-24,ss (fraction of dose excreted unchanged in urine over 24 h post-dose at steady-state) and dose-normalized AUCtau,ss and Cmax,ss. Pharmacokinetics were also compared between asthma and COPD, incorporating data from 3 COPD Phase II/III clinical trials. Tiotropium pharmacokinetics in asthma were dose-proportional up to 5 µg dosed once daily. The following factors showed no statistically significant effects on tiotropium systemic exposure in asthma based on analysis of geometric mean ratios and 90% confidence intervals: age, asthma severity, lung function, reversibility testing, allergy status, smoking history, geographical region, and posology (5 µg once daily or 2.5 µg twice daily via Respimat®). Asian patients showed a moderately but significantly higher systemic exposure compared to White or Black patients. However, no differences in safety by race were observed. Total systemic exposure (AUCtau,ss) was similar between asthma and COPD, but Cmax,ss was 52% lower in asthma patients compared to COPD. It is concluded that in asthma, patient characteristics have no relevant effect on tiotropium systemic exposure. Since systemic exposure to inhaled drugs is an indicator of safety, the lower Cmax,ss compared to COPD is not considered a concern for tiotropium therapy of asthma.

Pharmacokinetic Bioequivalence of Two Inhaled Tiotropium Bromide Formulations in Healthy Volunteers.

BACKGROUND AND OBJECTIVE: A novel tiotropium bromide monodose capsule dry powder inhaler (DPI) formulation and device have been developed. The formulation was based on a spray-dried matrix that enhances the aerosolizaton properties, allowing a less active tiotropium metered dose (13 µg/capsule) while maintaining the same delivered dose (10 µg/actuation). This study describes the pharmacokinetic bioequivalence to the reference product. METHODS: This randomized, two-stage, crossover, semi-replicate (three-way) study was performed in healthy volunteers. In each study period, subjects received a single dose of two capsules (20 µg delivered dose) of the study medication, separated by a 14-day washout period: tiotropium 10 µg delivered dose (Laboratorios Liconsa, Spain) and Spiriva HandiHaler(®) (Boehringer Ingelheim Pharma GmbH & Co KG, Germany). Blood samples were obtained up to 48 h post-dose to evaluate the comparative bioavailability. Tiotropium was measured in plasma by means of dual stage liquid-liquid extraction followed by the two-dimensional ultra-high performance liquid chromatography sensitive sub-pg/mL bioanalytical method. The main pharmacokinetic parameters were maximum plasma concentration (C max), area under the concentration-time curve (AUC) from time zero hours to the last observed concentration at time t (AUC t ), and AUC from time zero hours to 30 min (AUC0.5). Bioequivalence was accepted if the 90.20 % confidence interval (CI) for the ratio test/reference of the primary pharmacokinetic parameters lay within the acceptance range of 80-125 %. Safety assessment was a secondary endpoint. RESULTS: A total of 30 subjects were randomized and bioequivalence was demonstrated for all primary pharmacokinetic parameters: C max (CI 87.26-106.60 %), AUC t (CI 101.33-111.64 %), and AUC0.5 (CI 97.95-113.49 %). Both study treatments were well tolerated (four non-serious adverse events [AEs] were reported in four subjects: one AE before any product administration, two AEs after test product administration; and one AE after reference product administration). CONCLUSIONS: Both products containing tiotropium 10 µg delivered-dose DPI were bioequivalent and showed good tolerability and a similar safety profile.

Tiotropium Respimat(®) Versus HandiHaler(®): Comparison of Bronchodilator Efficacy of Various Doses in Clinical Trials.

INTRODUCTION: The long-acting muscarinic antagonist tiotropium bromide is approved in many countries as maintenance therapy for chronic obstructive pulmonary disease (COPD). Tiotropium is available as a dry-powder formulation delivered via HandiHaler(®) (18 µg once daily) and is now also approved as an aqueous solution delivered via the Respimat(®) Soft Mist™ Inhaler (5 µg once daily, 2 puffs of 2.5 µg). Several studies have compared the efficacy of tiotropium HandiHaler (18 µg once daily) with different doses of Respimat. We aimed to compare available bronchodilator efficacy data of once-daily Respimat 1.25, 2.5, 5, 10, 20 µg, and HandiHaler 18 µg to investigate which dose of tiotropium delivered by Respimat is the closest match to tiotropium HandiHaler. METHODS: Evaluation of six clinical trials (duration from 3 weeks to 2-3 years) that included lung function measures (trough forced expiratory volume in 1 s and trough forced vital capacity) as key outcomes. RESULTS: In the six trials, bronchodilator efficacy of Respimat 5 µg and HandiHaler 18 µg was similar; however, reduced bronchodilator efficacy was observed with lower doses of Respimat (1.25 and 2.5 µg). CONCLUSION: These findings support the use of the marketed once-daily dose of Respimat 5 µg for the maintenance treatment of patients with COPD. FUNDING: Boehringer Ingelheim.

Tiotropium/Olodaterol: A Review in COPD.

Tiotropium/olodaterol (Stiolto™ Respimat®, Spiolto™ Respimat®) is a fixed-dose combination of the long-acting antimuscarinic agent tiotropium bromide (hereafter referred to as tiotropium) and the long-acting ß2-adrenoreceptor agonist olodaterol delivered via the Respimat® Soft Mist™ inhaler. It is indicated for the maintenance treatment of airflow obstruction in adults with COPD. Several randomized, phase III studies of 6-52 weeks' duration evaluated the efficacy of once-daily tiotropium/olodaterol in patients with GOLD stage 2-3 or 2-4 COPD. Tiotropium/olodaterol maintenance therapy improved lung function to a greater extent than the individual components or placebo and provided clinically meaningful improvements in health-related quality of life and dyspnoea in 12- and 52-week studies. Tiotropium/olodaterol consistently improved 24-h lung function in 6-week studies, providing greater benefits than the monotherapies, placebo or twice-daily fixed-dose fluticasone propionate/salmeterol. Inspiratory capacity and exercise endurance were also improved with tiotropium/olodaterol following 6 or 12 weeks' treatment. The tolerability profile of tiotropium/olodaterol in the phase III studies was generally similar to that of the component monotherapies. The most common adverse events and serious adverse events during 52 weeks' therapy were respiratory in nature, with COPD exacerbation, unsurprisingly, reported most frequently with tiotropium/olodaterol and component monotherapies. Although additional data assessing the effect of tiotropium/olodaterol on exacerbations and comparative studies with other recommended therapies are needed to definitively position tiotropium/olodaterol, current evidence indicates that tiotropium/olodaterol is a useful treatment option for patients with COPD.

Combined bronchodilators (tiotropium plus olodaterol) for patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD), a respiratory disease characterized by a progressive decline in lung function, is considered to be a leading cause of morbidity and mortality. Long-acting inhaled bronchodilators, such as long-acting ß2 agonists (LABAs) or long-acting muscarinic antagonists (LAMAs), are the cornerstone of maintenance therapy for patients with moderate-to-very-severe COPD. For patients not sufficiently controlled on a single long-acting bronchodilator, a combination of different bronchodilators has shown a significant increase in lung function. Tiotropium, a once-daily dosing LAMA, demonstrated sustained improvements in lung function as well as improved health-related quality of life, reduced exacerbations, and increased survival without altering the rate of decline in the mean forced expiratory volume in 1 second (FEV1) with fairly tolerable side effects. Olodaterol is a once-daily dosing LABA that has proven to be effective in improving lung function, reducing rescue medication use, and improving dyspnea and health-related quality of life, as well as improving exercise endurance with an acceptable safety profile. The combination of olodaterol and tiotropium provided additional improvements in lung function greater than monotherapy with each drug alone. Several well-designed randomized trials confirmed that the synergistic effect of both drugs in combination was able to improve lung function and health-related quality of life without a significant increase in adverse effects. The objective of this paper is to review available evidence on the clinical efficacy and safety of tiotropium, olodaterol, and their combination in patients with COPD.

In Vitro, Pharmacokinetic, Pharmacodynamic, and Safety Comparisons of Single and Combined Administration of Tiotropium and Salmeterol in COPD Patients Using Different Dry Powder Inhalers.

In vitro Andersen cascade impactor-sized mass (ISM) and aerodynamic fine particle mass (FPM) <5 µm for tiotropium and salmeterol combined in a novel inhalation powder formulation containing 7.5 µg tiotropium/25 µg salmeterol (TSHH) were similar (within ±15%) to reference products containing 18 µg of tiotropium (Spiriva® HandiHaler®) (TioHH) and 50 µg of salmeterol (Serevent® Diskus®) (SalD). The pharmacokinetics (PK), pharmacodynamics, safety, and tolerability of the novel fixed-dose TSHH formulation administered once daily was compared with the single-agent therapies TioHH (once daily [qd]) and SalD (twice daily [bid]) and with the jointly administered combination of TioHH (qd) plus SalD (bid) in a randomized, 22-week, open-label, four-way crossover study in 50 patients with chronic obstructive pulmonary disease (COPD). For tiotropium, TSHH and TioHH were bioequivalent based on mean steady-state plasma area under the plasma concentration-time curves (AUC), while the urinary excretion amount was higher for TSHH and not bioequivalent to TioHH. Tiotropium peak plasma concentrations at steady state (C max,ss) were 40% higher with TSHH. For salmeterol, substantial differences were observed in plasma AUCs and Cmax,ss. No significant differences in 8-h forced expiratory volume in 1 s or forced vital capacity were detected for the TSHH (qd) against the combination of TioHH (qd) with SalD (bid). Maintenance therapy with tiotropium plus salmeterol as TSHH or as the jointly administered reference products is superior to either agent alone, safe, and well tolerated in COPD patients. In vitro results were not predictive of clinical PK findings for both tiotropium and salmeterol for the TSHH dry powder inhaler product.

Safety, tolerability, and plasma exposure of tiotropium Respimat® in children and adults with cystic fibrosis.

BACKGROUND: People with cystic fibrosis (CF) suffer from chronic lung disease that is often treated with a bronchodilator. This trial evaluated the pharmacokinetics, safety, and tolerability of single and multiple doses of tiotropium inhaled via the Respimat® Soft Mist™ Inhaler in patients with CF. METHODS: Patients received a single dose (placebo, 2.5 µg, 5 µg, or 10 µg) and/or multiple doses (placebo, 2.5 µg, or 5 µg) of tiotropium daily for 28 days. RESULTS: Ninety-two patients, aged 5-57 years, were treated. All doses showed a satisfactory safety profile for adverse events, vital signs, laboratory evaluations, and physical examination. At steady-state, peak exposure to tiotropium was comparable between adult patients with CF and patients with chronic obstructive pulmonary disease. CONCLUSIONS: Tiotropium 2.5 µg or 5 µg inhaled via the Respimat® Soft Mist™ Inhaler once daily was well tolerated in patients with CF.

Comparative study on pharmacokinetics of a series of anticholinergics, atropine, anisodamine, anisodine, scopolamine and tiotropium in rats.

The compound series of traditional anticholinergics [atropine (Atr), anisodamine (Ani), anisodine (AT3), and scopolamine (Sco)], naturally occurring belladonna alkaloid, have been approved for numerous therapeutic uses since 1970s. Tiotropium, a novel M receptor antagonist for the treatment of chronic obstructive pulmonary disease, was structurally modified based on atropine-like drugs. Clinical phenomena suggested that the changes of substituent group were related to the pharmacokinetic and pharmacodynamic characteristics of the agents. In an attempt to compare the pharmacokinetics of the series of anticholinergics and investigate the subsets motivating selective anticholinergic potencies, a sensitive LC-MS/MS method was established to analyze the differences of pharmacokinetic parameters. In this paper, we determined the pharmacokinetics of atropine, anisodamine, anisodine, scopolamine, and tiotropium after i.v. and i.g. single dose administration. After i.v. administration, the maximum drug plasma concentrations (C max) of Atr, Ani, AT3, and Sco were 274.25 ± 53.66, 267.50 ± 33.16, 340.50 ± 44.52, and 483.75 ± 78.13 ng/mL. Tiotropium had a slightly higher area under the curve with a significant increase of C max value. Because of their partial solubility, Atr, Ani, AT3, and Sco had different bioavailability in rats of 21.62, 10.78, 80.45 and 2.52 %, respectively. Following i.g. administration of tiotropium, the C max value below 20 ng/mL revealed the very low oral absorption. The urinary excretion rates of Atr, Ani, AT3, Sco and tiotropium were 11.33, 54.86, 32.67, 8.69 and 73.91 %. This work provided relatively comprehensive preclinical data on the series of anticholinergics, which may be used to explain the clinical adverse effects and applications.