Effect of the inhaled PDE4 inhibitor CHF6001 on biomarkers of inflammation in COPD

Background CHF6001 is a novel inhaled phosphodiesterase-4 inhibitor. This Phase IIa study assessed the effects of CHF6001 on markers of inflammation in induced sputum and blood in patients with chronic obstructive pulmonary disease (COPD). Methods This was a multicentre, three-period (each 32 days), three-way, placebo-controlled, double-blind, complete-block crossover study. Eligible patients had COPD, chronic bronchitis, and were receiving inhaled triple therapy for ≥2 months. Patients received CHF6001 800 or 1600 μg, or matching placebo twice daily via multi-dose dry-powder inhaler (NEXThaler). Induced sputum was collected pre-dose on Day 1, and post-dose on Days 20, 26 and 32. Blood was sampled pre-dose on Day 1, and pre- and post-dose on Day 32. Results Of 61 randomised patients, 54 (88.5%) completed the study. There were no significant differences between groups for overall sputum cell count, or absolute numbers of neutrophils, eosinophils or lymphocytes. CHF6001 800 μg significantly decreased the absolute number and percentage of macrophages vs placebo. In sputum, compared with placebo both CHF6001 doses significantly decreased leukotriene B4, C-X-C motif chemokine ligand 8, macrophage inflammatory protein 1β, matrix metalloproteinase 9, and tumour necrosis factor α (TNFα). In blood, both CHF6001 doses significantly decreased serum surfactant protein D vs placebo. CHF6001 1600 μg significantly decreased TNFα ex-vivo (after incubation with lipopolysaccharide). Conclusion The data from this study show that CHF6001 inhaled twice daily has anti-inflammatory effects in the lungs of patients with COPD already treated with triple inhaled therapy. Trial registration The study is registered on ClinicalTrials.gov (NCT03004417). Electronic supplementary material The online version of this article (10.1186/s12931-019-1142-7) contains supplementary material, which is available to authorized users.

• Ex-vivo lipopolysaccharide (LPS) stimulated TNFα: 1.5 mL of blood collected into sodium heparin tubes was stimulated with 30 µL of LPS 5 µg/mL, incubated at 37C for 23 h, centrifuged at 2000 g for 10 min at 4C and plasma transferred to the central laboratory at -60 to -90C for analysis.
• Fibrinogen and MMP-3 and -9: Blood was collected into lithium heparin tubes, centrifuged at 2500 g for 15 min at 4C, and plasma transferred to the central laboratory at -60 to -90C for analysis.
Levels of biomarkers were assessed as follows: • Fibrinogen: K-Assay Fibrinogen kit, automated on the Beckman AU640.
Validation procedures again followed those outlined for the analysis of biomarkers above. All the methods were found to be accurate and precise and deemed suitable for measuring concentrations of biomarkers in human serum or plasma from regulatory studies.
Blood for the pharmacokinetic analysis was collected pre-dose and at 30 min, and 1, 1.5, 2, 3, 4, 6, 8 and 12 h post-dose on Day 32 of each treatment period. Samples were centrifuged at 2000 g for 15 min at 4C, with the resulting plasma shipped to the central laboratory at -60 Effect of CHF6001 on inflammatory biomarkers -Additional file 1 Page 6 of 17 to -90C. The plasma concentration of CHF6001 was evaluated using a LC-MS/MS method following solid phase extraction. Validation procedures again followed those outlined for the analysis of biomarkers above. The bioanalytical method was found to be linear over the calibration range of 10 to 20,000 pg/mL. The precision and accuracy of the method was found to be within the target limits of within 20% at the lower limit of quantification 10 pg/mL and within 15% at all other concentrations.
The recovery of CHF6001 from human plasma was consistent across the analytical range, with no significant matrix effects. CHF6001 was found to be stable in human plasma stored for up to 24 h at the sample processing temperature (nominally +20C), after storage for 184 days in a freezer (nominally -80C) and after four freeze-thaw cycles at nominally -80C / +20C. Therefore the method was considered to be suitable for measuring concentrations of CHF6001 in human plasma samples from regulatory studies.

Ethics committees
The study was approved by two centralised ethics committees on behalf of the sites:

Inclusion Criteria
Patients had to meet all the following inclusion criteria to be eligible for enrolment into the study: 1. Written informed consent obtained prior to any study-related procedures; 2. Male or female aged ≥40 years; 3. A female was eligible to enter the study if she was of non-childbearing potential, i.e. physiologically incapable of becoming pregnant (e.g. postmenopausal women defined as being amenorrhoeic for ≥12 consecutive months without an alternative medical cause. If indicated, as per Investigator's request, post-menopausal status was confirmed by analysis of follicle-stimulating hormone levels, according to local laboratory ranges) or women permanently sterilised (e.g. bilateral oophorectomy, hysterectomy or bilateral salpingectomy). Women physiologically capable of becoming pregnant (i.e. women of childbearing potential) were eligible to enter the study if they had a negative pregnancy test at screening and agreed to use one or more of the following highly effective contraceptive measures: a. Placement of an intrauterine device or intrauterine hormone Reliable contraception had to be maintained throughout the study. of salbutamol via pressurised metered-dose inhaler pMDI. If this criterion was not met at screening, the test was repeated once before the randomisation visit; 8. Patients must have been receiving daily maintenance with triple therapy (inhaled corticosteroid [ICS] plus long-acting muscarinic antagonist [LAMA] plus long-acting β2-agonist [LABA]) at a stable dose and dosing regimen for at least two months prior to screening; 9. A history of chronic bronchitis defined as chronic cough and sputum production for more than three months per year for two or more years and known as a 'spontaneous sputum producer'; 10. At screening, patients must have been able to produce an adequate induced sputum sample defined as a load of at least 300 mg with a viability factor of not less than 70% (with less than 30% epithelial cells) and a neutrophil % differential count of at least 60%. The patients may have been re-challenged once, if the first sputum sample did not meet these criteria; 11. Patients must have been symptomatic at screening, defined as having a COPD Assessment Test score ≥10; 12. Patients had to be able to be trained to use the DPI inhalers (NEXThaler ® ) correctly and to generate sufficient peak inspiratory flow (PIF) (at least 40 L/minute) using the In-Check Dial ® device; 13. A cooperative attitude and ability to perform the required outcome measurements (e.g. spirometry testing, induced sputum, and other analyses).

Exclusion Criteria
The presence of any of the following excluded a patient from study enrolment: 7. Patients participating in a pulmonary rehabilitation program or completing such a program within the last six weeks prior to study entry; 8. Patients with known respiratory disorders other than COPD that in the Investigator's opinion would affect efficacy and safety evaluation or place the patient at risk. This included, but was not limited to, known α-1 antitrypsin deficiency, active tuberculosis, bronchiectasis, sarcoidosis, lung fibrosis, pulmonary hypertension and interstitial lung disease; 9. Patients with lung cancer or a history of lung cancer; 10. Patients with active cancer or a history of cancer (other than lung) with less than five years disease-free survival time (whether there was evidence of local recurrence or metastases or not). Localised carcinoma (e.g. basal cell carcinoma [without metastases], in situ carcinoma of the cervix adequately treated) was acceptable; 11. Patients with a known history of hypersensitivity to β2-agonists, PDE4 inhibitors or any of the excipients contained in any of the formulations used in the study; 12. Patients with a diagnosis of depression associated with suicidal ideation or behaviour or with a diagnosis of generalised anxiety disorder that in the Investigator's opinion would place the patient at risk; 13. Patients who had known history of clinically significant (CS) cardiovascular conditions such as, but not limited to, unstable or acute ischemic heart disease within one year prior to study entry, New York Heart Association Class III/IV heart failure, known history of sustained and non-sustained cardiac arrhythmias or history of atrial fibrillation diagnosed in the last 6 months prior to study entry and not controlled with therapy rate control strategy; 20. Current or chronic history of liver disease, or known hepatic or biliary abnormalities (except for Gilbert's syndrome or asymptomatic gallstones); 21. Patients receiving treatment with any drug known to have a well-defined potential for hepatotoxicity (e.g. isoniazid, nimesulide, ketoconazole) within the previous three months prior to study entry and during the screening period; 22. Patients who experienced excessive weight loss recently (which could not be explained by the natural course of COPD or known background conditions); 23. Patients with a history of alcohol abuse and/or substance/drug abuse within 12 months prior to the screening visit; 24. Patients that received any other investigational drug within the preceding 30 days (60 days for biologics), or a longer and more appropriate time as determined by the Investigator (e.g. approximately five half-lives of the previous investigational drug).

Non-permitted COPD concomitant medication (and duration prior to screening)
1. Depot corticosteroids (two months);

Gastrointestinal adverse events
The adverse event system order class 'gastrointestinal disorders' includes reports of the following preferred terms: abdominal pain, aphthous ulcer, breath odour, dental caries, constipation, diarrhoea, dry mouth, dry lip, enteritis, inguinal hernia, nausea, paraesthesia oral, toothache and vomiting.