This Article Abstract Full Text (PDF) Free Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Article Archive Download to citation manager Citing Articles Citing Articles via ISI Web of Science (11) Citing Articles via Google Scholar Google Scholar Articles by Grasemann, H. Articles by Ratjen, F. Search for Related Content PubMed PubMed Citation Articles by Grasemann, H. Articles by Ratjen, F.

Inhalation of Moli1901 in Patients With Cystic Fibrosis^*

^* From the Children’s Hospital (Drs. Grasemann, Stehling, and Ratjen), University of Duisburg-Essen, Essen, Germany; AOP Orphan Pharmaceuticals AG (Drs. Brunar and Widmann), Vienna, Austria; Lantibio Inc (Ms. Laliberte and Dr. Molina), Chapel Hill, NC; and the Institute of General and Environmental Hygiene (Dr. Döring), University of Tübingen, Tübingen, Germany.

Correspondence to: Hartmut Grasemann, MD, The Hospital for Sick Children, 555 University Ave, Toronto, ON, Canada M5G 1X8, e-mail: hartmut.grasemann{at}sickkids.ca

Abstract

Background: In cystic fibrosis (CF) patients, the absence or dysfunction of the chloride channel CF transmembrane conductance regulator (CFTR) results in reduced chloride ion transport in respiratory epithelial cells. Moli1901 stimulates an alternative chloride channel and may thus compensate for the CFTR deficiency in the airway epithelium of CF patients.

Methods: A phase II, placebo-controlled, double-blinded, single-center, multiple (5 consecutive days), rising-dose (daily dose, 0.5, 1.5, or 2.5 mg of Moli1901) study was conducted to investigate the safety and tolerability of multiple doses of aerosolized inhaled Moli1901 in 24 patients with CF and stable lung disease.

Results: Moli1901 was well tolerated in all but one CF patient, in whom a transient significant decrease in FEV₁ developed following inhalation, which resolved spontaneously, and in a second patient in whom transient throat numbness developed during drug inhalation. A significant improvement of FEV₁ was observed in the group receiving treatment with 2.5 mg/d Moli1901 compared to the group receiving placebo (p = 0.01 [Wilcoxon test]). Moli1901 was not detected in the plasma of the highest dose group.

Conclusions: The inhalation of Moli1901 up to a total cumulative dose of 12.5 mg appears to be safe in adult patients with CF. In addition, Moli1901 had a sustained beneficial effect on pulmonary function, which supports further studies of its efficacy in CF patients.

Key Words: absorption • antibiotics • clinical phase II trial • cystic fibrosis • inhalation drug administration • pediatric

Cystic fibrosis (CF) is characterized by abnormal chloride ion transport in epithelial cells. This results in altered sodium and water absorption from the airway surface liquid (ASL), a reduced ASL layer, and impaired airway clearance.¹

Moli1901 (duramycin) is a stable 19-residue polycyclic peptide that is derived from Streptomyces cinnamoneum. It interacts with phospholipids that are present in plasma and organelle membranes,² where it activates an alternative chloride channel by elevating intracellular calcium levels.³⁴ Moli1901 has been shown to increase chloride transport and fluid secretions when applied to the airway epithelium in vitro⁵⁶ and increases chloride permeability in the nasal epithelium of healthy individuals and patients with CF.⁷ In addition, animal experiments have shown that Moli1901 increased the volume of ASL, was slowly absorbed into and excreted from the lung, and did not accumulate in other tissues.⁵⁸⁹

An increased activity of the alternative chloride epithelial channels in the lower airways, observed after the inhalation of Moli1901, may compensate for reduced or absent CF transmembrane conductance regulator (CFTR) function and potentially improve clinical status in CF patients. We report here the results of a phase II, placebo-controlled, double-blind, single-center study on the safety and tolerability of multiple, rising doses of aerosolized Moli1901 in patients with CF and stable pulmonary disease. This study not only shows that the inhalation of Moli1901 appears to be safe in CF patients but also suggest a beneficial effect on pulmonary function at higher doses.

Materials and Methods

Study Design
The study (Moli1901–008) was designed as a phase II, placebo-controlled, double-blinded, single center, multiple, rising-dose study. The study was approved by the Institutional Review Board of the University of Duisburg-Essen, Germany. Written informed consent was obtained from each CF patient and/or their parents.

Inclusion and Exclusion Criteria
Patients were eligible for participation if they were 16 years of age, had a confirmed diagnosis of CF (ie, positive sweat chloride value of 60 mEq/L and/or two identified CFTR gene mutations), and were in stable clinical condition with a baseline FEV₁ of > 60% predicted and oxygen saturation of > 90% on room air. Exclusion criteria were Burkholderia cepacia infection; the presence of allergic bronchopulmonary aspergillosis; changes in routine maintenance therapy within 4 weeks of undergoing screening; clinically significant liver, renal, cardiac, neurologic, or hematologic disease; poorly controlled diabetes mellitus; a history of alcohol or drug abuse; as well as participation in an investigational study within 4 weeks of undergoing screening. Women of child-bearing potential refusing to use effective contraception or being pregnant or lactating were also not eligible for the study. Participating women were advised not to become pregnant for 6 months after the study.

Drug Formulation
The study drug was provided as either a sterile Moli1901 solution for inhalation (Moli1901) in a concentration of 0.5 mg/mL in a 0.9% normal saline solution or placebo (0.9% normal saline solution) in 5-mL single-use vials. Moli1901 and placebo were shipped directly to the hospital pharmacy at the study site and were stored at a temperature of 2 to 8°C. The 0.1 and 0.3 mg/mL Moli1901 solutions were diluted from the 0.5 mg/mL solution and blinded to the investigator. Moli1901 or placebo was placed in a nebulizer canister (PARI LC Plus; PARI; Starnberg, Germany) immediately prior to administration to the patient on the treatment day.

Study Subjects
Three cohorts of eight subjects each, with six subjects receiving Moli1901 and two subjects receiving placebo, were studied. Each subject within a cohort inhaled 5-mL doses of the 0.1, 0.3, or 0.5 mg/mL concentration of Moli1901 or placebo once daily for 5 consecutive days. Subjects in the first cohort received a cumulative dose of 2.5 mg of Moli1901 (daily dose, 0.5 mg; concentration, 0.1 mg/mL x 5 mL). This cumulative dose is equivalent to the highest single dose of Moli1901 that was shown to be safe and tolerated in CF patients in an earlier study.¹⁰ Subjects in the second cohort received a cumulative dose of Moli1901 of 7.5 mg (daily dose, 1.5 mg; concentration, 0.3 mg/mL x 5 mL), and subjects in the third cohort received a cumulative dose of 12.5 mg (daily dose 2.5 mg; concentration, 0.5 mg/mL x 5 mL). A Data Safety Monitoring Board determined safety before the start of the second and third cohorts.

Safety and tolerability were assessed by the spirometric evaluation of pulmonary function, vital signs, peripheral oxygen saturation, physical examination, clinical laboratory tests, ECG, and the monitoring of adverse events (AEs). Pretreatment and posttreatment (ie, 30 min, 60 min, 2 h, and 4 h) pulmonary function evaluations included FEV₁, FVC, and forced expiratory flow to 25 to 75% of FVC, which were performed according to American Thoracic Society guidelines.¹¹ Patients receiving regular inhaled bronchodilator therapy were asked to omit the morning dose on study days. Exercise tolerance was determined by 3-min step test,¹² and quality of life was determined by revised CF questionnaire.¹³

A severe AE was defined a priori as a decline in FEV₁ of 20% from baseline accompanied by symptoms (eg, cough, wheeze, chest tightness, or shortness of breath), a decrease in oxyhemoglobin saturation to < 90% or a decline of 8% from baseline that requires therapeutic intervention, or changes in clinical laboratory test results, ECG, or other safety parameters judged to be clinically significant.

Laboratory Evaluations
Blood samples were collected at screening and on day 5 postdose. Blood samples were also collected on days 12 (± 2), 19 (± 2), and 26 (± 2), if clinically significant abnormal findings had been noted in prior samples. Safety laboratory assessments of the specimen included chemistry (ie, measurement of electrolyte, bicarbonate, creatinine, BUN, lactate dehydrogenase, total bilirubin, gamma glutamyl transferase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, albumin, and glucose levels), hematology (ie, CBC count and differential count, hematocrit, hemoglobin, mean cell volume, and activated partial thromboplastin time [aPTT]), and serum pregnancy tests on days 1 and 26 (women only).

Moli1901 plasma concentrations were determined at screening and on day 5 (posttreatment) in the group receiving treatment with 0.5 mg/mL Moli1901 only. Moli1901 concentrations were measured (liquid chromatography/mass spectrometry technology; Pharm-analyt Labor GmbH; Baden, Austria) at a lower limit of detection of < 1 ng/mL. Urine analysis included specific gravity, glucose, protein, leukocytes, erythrocytes, and casts.

Statistical Analysis
Participant demographics of the three cohorts were analyzed and compared with regard to gender, age, FEV₁, concomitant treatment, and quality of life at baseline. All enrolled subjects were included in efficacy and safety analyses according to the intention-to-treat principle.

Descriptive statistics were used to summarize safety parameters including pulmonary function, pulse oximetry, vital signs, physical examination findings, ECG, and laboratory test results. Changes from baseline were compared among all four groups using a Kruskal-Wallis test. If this overall test was significant, pairwise comparisons were conducted between the placebo group and each dosing group (Wilcoxon tests). Changes in laboratory parameters within each group were evaluated using a Wilcoxon test for paired observations. The results of these tests were interpreted explorative. Data management and statistical analyses were conducted by Assign Data Management and Biostatistics GmbH, Innsbruck, Austria.

Results

Demographic and Baseline Characteristics
The majority of CF patients were CFTR F508 homozygous (n = 14), compound heterozygous (n = 6), or F508/unknown (n = 3). Demographics and baseline characteristics of the study groups are given in Table 1 . The median screening FEV₁ was 90% predicted (range, 58 to 121% predicted). A nonsignificant tendency toward lower values in the group receiving a dose of 1.5 mg/d was observed (p = 0.2). Significant differences were seen for screening pulse oximetry (median, 96.5%; range, 95 to 100%; p = 0.034), with the highest values in the group receiving a dose of 2.5 mg/d (median, 99.5%), and for body mass index (p = 0.039). The findings of the physical examination screening and 12-lead ECG were normal in all subjects, and the body temperature, respiratory rate, heart rate, and BP were comparable among the groups, as were the results of the baseline exercise tolerance tests.

Statistically significant changes in laboratory parameters from day 1 to day 5 were observed for the total study population; however, the changes were not different between treatment groups. Changes included increased WBC count (median change, 1.64 cells/nL; range, –1.6 to 5.6 cells/nL; p = 0.0006), monocytes (median change, –1.2%; range, –4.4 to 4.6 cells/nL; p = 0.014), neutrophils (median change, 5.0%; range, –14.0 to 28.0 cells/nL; p = 0.039), and prolonged aPTT (median change, 1.75 s; range, –2.0 to 4.8 s; p = 0.0005 [Wilcoxon test]). Changes from day 1 to day 26 were significant only for aPTT for the total study sample (median change, 1.3 s; range, 0.1 to 6.2; p = 0.004 [Wilcoxon test]); again, no significant differences in laboratory parameters were found among the treatment groups. No statistically significant changes in CF questionnaire score were observed from screening to the end of treatment for any treatment group and the total study sample.

Efficacy
The median FEV₁ change from day 1 to day 5 was –2.5% in the placebo group, 2% in the 0.5 mg/d treatment group, 6% in the 1.5 mg/d treatment group, and 7% in the 2.5 mg/d treatment group. The differences among treatment groups were statistically significant (p = 0.027 [Kruskal-Wallis test]). Post hoc paired comparisons demonstrated a significant difference between the 2.5 mg/d treatment group and the placebo group (p = 0.01 [Wilcoxon test]) but not for the other treatment groups (Fig 1 , Table 3 ). The median FEV₁ change from day 1 to day 26 amounted to –1% in the placebo group, –8.5% in the 0.5 mg/d treatment group, 2% in the 1.5 mg/d treatment group, and 8% in the 2.5 mg/d treatment group. The differences among treatment groups were statistically significant (p = 0.023 [Kruskal-Wallis test]), but were not significant in post hoc comparisons.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 1.. Change in FEV1 from baseline in CF patients treated with Moli 1901 or placebo. Data are shown as mean ± SEM.

Median forced expiratory flow to 25 to 75% of FVC decreased 3.5% in the placebo group and 0.5% in the 0.5 mg/d treatment group, and increased 7% in the 1.5 mg/d treatment group and 5% in the 2.5 mg/d treatment group at day 5. These changes were not statistically significant. Changes in median forced expiratory flow to 25 to 75% of FVC from days 1 to 26 were –11% for the placebo group and –12% for the 0.5 mg/d treatment group, but 1% for the 1.5 and 2.5 mg/d treatment groups, respectively (p = 0.053 [Kruskal-Wallis test]).

Discussion

The inhalation of nebulized Moli1901 was well-tolerated by the CF patients included in this phase II trial. Although a transient significant decrease in FEV₁ following the inhalation of Moli1901 occurred in one patient in the 1.5 mg/d treatment group, this resolved spontaneously. While this study was not primarily powered to assess efficacy, the present data additionally suggest that inhaled Moli1901 may result in a dose-dependent increase in pulmonary function in patients with CF.

The intranasal application of Moli1901 in single doses with concentrations up to 10 µmol/L was previously found⁷ to be safe and well-tolerated in healthy volunteers and CF patients. Chloride secretion was induced in both healthy and CF nasal epithelium exposed to Moli1901 in this study, although the response was more variable in CF patients.⁷ Previous studies had shown that the half-life of Moli1901 was 2 months (64 days) in rat lungs.¹⁴ The prolonged presence of Moli1901 in dog lungs after inhalation resulted in a persistent drug effect of at least 1 week⁶⁹; however, the response to nasal inhalation of Moli1901 in the CF participants was relatively short.⁷ The accelerated turnover of the CF airway epithelium resulting in clearance of the drug from the lung may be one factor contributing to a reduced half-life of the drug in CF patients. Therefore, better results may be obtained with repeated inhalations of Moli1901. The results from our study suggest that the beneficial effect of a 2.5-mg daily dose of Moli1901 was sustained, since FEV₁ was still increased at 26 days in this group (Fig 1). Using a method of detection at a lower limit of quantification of < 1 ng/mL Moli1901 in plasma, Moli1901 appears not to enter the systemic circulation, as is shown by the lack of Moli1901 in the plasma of the highest dose group. Although it is unclear whether repeated inhalations of the drug result in sustained action, it is worth mentioning that Moli1901 is minimally absorbed from the lungs of rats and dogs, and is excreted in the feces, presumably as the result of mucociliary clearance and swallowing.⁹¹⁴ This mode of excretion may allow prolonged clinical activity in the lungs after repeated inhalations without relevant systemic side effects.

The stimulation of alternative chloride channels in the airway epithelium may be sufficient to bypass absent or dysfunctional CFTR chloride conductance in the airways of CF patients. In vivo studies¹⁵ have shown that calcium-activated, non-CFTR chloride conductance is present in the human CF epithelium but is inactive unless intracellular calcium is increased. Moli1901, when applied to the apical surface of the airway epithelium, increased intracellular calcium concentration from both extracellular fluid as well as intracellular stores.⁴ This resulted in an increase of short-circuit current and net chloride secretion as has been demonstrated in the canine tracheal epithelium as well as the healthy human and human CF epithelium.²³⁶ The response to Moli1901 was unaffected by the pretreatment of tissues with amiloride (10⁴ mol/L) or cyclic adenosine monophosphate (10³ mol/L), suggesting that the effect may not involve Na⁺ channels or CFTR.⁷ In addition to effecting an increase in chloride secretion, Moli1901 may also have a positive effect on ASL volume, as suggested by experiments in dogs⁵⁶ in which Moli1901 was administered by aerosol.

Decreased ASL is a hallmark in CF patients, and the concept of restoring ASL volume is currently being addressed in other clinical studies. These agents being studied include hyperosmotic agents, such as mannitol, administered via inhalation¹⁶ or hypertonic saline solution,¹⁷ inhibitors of the epithelial Na⁺ current,¹⁸ and P2Y₂ receptor agonists.¹⁹ While sufficient evidence supports the use of hypertonic saline solution in CF patients,¹⁷ the effects of the other treatment approaches are currently unclear. Subsequent studies will delineate which of these therapies are best suited to improve airway surface hydration and to restore mucociliary transport in patients with CF.

Results from this phase II study show that the repeated inhalation of Moli1901, an activator of alternative chloride channels, appears to be safe in adolescent and adult CF patients with mild lung disease. The lack of detectable Moli1901 levels substantiates the likelihood that there is little or no systemic absorption of inhaled Moli1901. The data further suggest a beneficial effect of repeated inhalations of a 2.5-mg daily dose of Moli1901 for 5 days on pulmonary function in these patients. Larger clinical trials are needed to study the safety and efficacy of longer treatment regimens with aerosolized inhaled Moli1901 in CF patients.

Acknowledgements

The authors wish to acknowledge the valuable support of this study by Christine Bauer, Manuela Groch, and Dr. Peter Tinschmann.

Footnotes

Abbreviations: AE = adverse event; aPTT = activated partial thromboplastin time; ASL = airway surface liquid; CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator

This work was performed at the Children’s Hospital, University of Duisburg-Essen, Essen, Germany.

Drs. Grasemann, Stehling, and Ratjen have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Dr. Molina is the founder and Chief Executive Officer of Lantibio Inc and holds equity in the company. Ms. Laliberte is a member of the management team of Lantibio Inc and holds equity in the company. Dr. Widmann is founder and Chief Executive Officer of AOP Orphan Pharmaceuticals AG, and holds equity in the company; Dr. Brunar is employed with AOP Orphan Pharmeceuticals AG. Consequently, both have a financial interest in the subject matter. Dr. Döring has received consultancy fees from AOP Orphan Pharmaceuticals AG.

Received for publication August 23, 2006. Accepted for publication December 23, 2006.

References

1. Ratjen, F, Döring, G (2003) Cystic fibrosis. Lancet 361,681-689[CrossRef][ISI][Medline]
2. Navarro, J, Chabot, J, Sherrill, K, et al Interaction of duramycin with artificial and natural membranes. Biochemistry 1985;24,4645-4650[CrossRef][Medline]
3. Cloutier, MM, Guernsey, L, Mattes, P, et al Duramycin enhances chloride secretion in airway epithelium. Am J Physiol 1990;259,C450-C454[ISI][Medline]
4. Cloutier, MM, Guernsey, L, Sha’afi, RI Duramycin increases intracellular calcium in airway epithelium. Membr Biochem 1993;10,107-118[ISI][Medline]
5. Molina y Vedia L, Stutts MJ, Boucher RC, et al. Method of treating retained pulmonary secretions. US patent 5 512 269; April 30, 1996
6. Henke, DC, Stutts, MJ, Boucher, RC, et al Bioelectric and fluid transport effects of the nonadecapeptide 2622U90 (duramycin) in normal human cystic fibrosis airway epithelium and canine airways [abstract].Pediatr Pulm 1998;17(suppl),238
7. Zeitlin, PL, Boyle, MP, Guggino, WB, et al A phase I trial of intranasal Moli1901 for cystic fibrosis. Chest 2004;125,143-149[Abstract/Free Full Text]
8. Rickert, DE, Dingley, K, Ubick, E, et al Determination of the tissue distribution and excretion by accelerator mass spectrometry of the nonadecapeptide 14C-Moli1901 in beagle dogs after intratracheal instillation. Chem Biol Interact 2005;155,55-61[CrossRef][ISI][Medline]
9. McNulty, MJ, Hutabarat, RH, Findlay, JWA, et al The disposition of the nonadecapeptide, duramycin, in the beagle dog after inhalation exposure. Proceedings of the Eighth North American Meeting of the International Society for the Study of Xenobiotics 1997;vol 12,123
10. Zeitlin, P, Brody, A, Waltz, D, et al A safety and pharmacodynamic assessment of inhaled administration of Moli1901 in subjects with cystic fibrosis. [abstract].Pediatr Pulmonol 2002;24(suppl),259
11. American Thoracic Society.. Standardization of spirometry: 1987 update; statement of the American Thoracic Society. Am Rev Respir Dis 1987;136,1285-1298[ISI][Medline]
12. Narang, I, Pike, S, Rosenthal, M, et al Three-minute step test to assess exercise capacity in children with cystic fibrosis with mild lung disease. Pediatr Pulmonol 2003;35,108-113[CrossRef][ISI][Medline]
13. Wenninger, K, Aussage, P, Wahn, U, et al German Cystic Fibrosis Questionnaire Study Group: the revised German Cystic Fibrosis Questionnaire: validation of a disease-specific health-related quality of life instrument. Qual Life Res 2003;12,77-85[CrossRef][ISI][Medline]
14. McNulty, MJ, Hutabarat, RH, Findlay, JWA, et al Pharmacokinetics and tissue distribution of the nonadecapeptide Moli1901 in rats and mice. Xenobiotica 2003;33,197-210[CrossRef][ISI][Medline]
15. Knowles, MR, Clarke, LL, Boucher, RC Extracellular ATP and UTP induce chloride secretion in nasal epithelia of cystic fibrosis patients and normal subjects in vivo. Chest 1992;101(suppl),60S-63S[Medline]
16. Robinson, M, Daviskas, E, Eberl, S, et al The effect of inhaled mannitol on bronchial mucus clearance in cystic fibrosis patients: a pilot study. Eur Respir J 1999;14,678-685[Abstract]
17. Elkins, MR, Robinson, M, Rose, BR, et al A controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis. N Engl J Med 2006;354,229-240[Abstract/Free Full Text]
18. Bowden, A, Morton, M, Stevens, L, et al Effect of the serine protease inhibitor AER 002 on epithelial sodium channel activity: a novel treatment for CF and COPD [abstract].Proc Am Thorac Soc 2006;3,A717
19. Deterding, R, Retsch-Bogart, G, Milgram, L, et al Safety and tolerability of denufosol tetrasodium inhalation solution, a novel P2Y2 receptor agonist: results of a phase 1/phase 2 multicenter study in mild to moderate cystic fibrosis. Pediatr Pulmonol 2005;39,339-348[CrossRef][ISI][Medline]

This Article Abstract Full Text (PDF) Free Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Article Archive Download to citation manager Citing Articles Citing Articles via ISI Web of Science (11) Citing Articles via Google Scholar Google Scholar Articles by Grasemann, H. Articles by Ratjen, F. Search for Related Content PubMed PubMed Citation Articles by Grasemann, H. Articles by Ratjen, F.