Severe AsthmaPhase 4NCT05001529
Flow Cytometry Analysis of Eosinophils in Severe Asthma Patients.
A Phase 4 study testing anti IL5 receptor antibodies in people with Severe Asthma, run by Scarlata, Simone, M.D. at 1 site in Italy.
Trial summary
Asthma is a heterogeneous disease, characterized by reversible airflow obstruction, airway hyperresponsiveness, and airway inflammation, in which 40% of patients exhibit eosinophil-driven pathobiology.The main treatment of asthma is the use of corticosteroid, whose use induces a reduction in eosinophils that is… Read more about this study
- Who is it for?
- Ages 18 years and older
- Conditions the study is for
- Severe Asthma
- What is being tested?
- anti IL5 receptor antibodies
- Could I receive a placebo?
- No, everyone receives an active treatment
- How many people
- 80 participants
- Where
- 1 siteItaly
- Study dates
- 18 Mar 2021to Mar 2023
- Phase
- Phase 4
- Are there results?
- None posted in the registry
Key requirements
Who can take part, as listed by the sponsor. Only the study team can confirm whether you are eligible, so take this list to your doctor.
- Ages 18 years and older
- All sexes
- No healthy volunteers
- Adult, Older adult
Who may be able to join
- proved diagnosis of severe, refractory eosinophilic asthma, according to GINA recommendations and International ERS/ATS guidelines;
- agreeing to participate this study and signing an informed consent.
Who cannot join
- current smoking habit (both tobacco and e-cigarettes),
- concomitant diseases requiring chronic administration of immunosuppressors, biologic medications or systemic corticosteroids for any disease other than asthma
- History of previous or concomitant acute or chronic disease known to directly or indirectly affect eosinophil count, both in a quantitative and qualitative manner (eosinophilic lung and gastrointestinal diseases, systemic vasculitis, allergic bronchopulmonary aspergillosis, parasitic infections, etc)
- COPD/Asthma overlap.
- Inability or denial to sign the informed consent.
Trial locations and contacts
1 site in 1 country.
Italy1 site, 1 recruiting
- Recruiting
Campus Bio Medico University and Teaching Hospital
Roma, 00136
Simone Scarlata, MD, +393939259912, s.scarlata@unicampus.it
About this study
Official title: Flow Cytometry Analysis of Eosinophils Activity and Membrane Receptors Expression in Severe Asthma Patients: Basal Characterization and Evaluation of Changes Induces by Biological Drugs.
Asthma is a heterogeneous disease, characterized by reversible airflow obstruction, airway hyperresponsiveness, and airway inflammation, in which 40% of patients exhibit eosinophil-driven pathobiology.The main treatment of asthma is the use of corticosteroid, whose use induces a reduction in eosinophils that is considered a strong predictor of response to treatment. Corticosteroids have remained the mainstay treatment of asthma and reduction in eosinophils has remained the unequivocal predictor of steroid response. The prevalence of asthma, which is expected to increase, it is about 300 million people worldwide. About 5-10% of asthma patients have severe disease, which is defined as asthma that requires high-dose inhaled corticosteroids (ICSs) plus a second controller to prevent it from becoming "uncontrolled" or which remains "uncontrolled" despite this therapy. Patients with severe disease have worse quality of life, and disproportionately high morbidity, mortality, and use of health care resources when compared with their peers with well-controlled disease.The pathophysiology of asthma is complex and heterogeneous between patients, as the disease itself; however, on the basis of immune system involvement, it is possible to define 2 subtypes - or endotypes- of asthma. These endotypes are named T2 (for type 2 cells) high or low, and are defined by the levels of expression of the T2 cytokines, IL-4, IL-5, and IL-13 produced by T helper 2 lymphocytes, and innate lymphoid cell-2.T2 high endotype patients display an increase in the number of blood and sputum eosinophils, and have a better response to the current available biological therapies , such as the administration of mepolizumab (anti IL-5 antibody). Eosinophilic asthma is associated to a more severe clinical phenotype,but patients with a T2 endotype respond better to biological therapies. The hypothesis of the present proposal is that the activation status of these cells, analyzed by the expression of activation markers, can be used to define a new, different, endotype, in which eosinophils, although quantitatively low or normal, are qualitatively more active and aggressive, and could therefore act as an indicator of the progression toward a T2 high endotype.Moreover, the investigators will verify whether a different expression of these molecules on eosinophil's surface might be associated with different clinical response to biologic medications.
Read the detailed description
Subject satisfying eligibility criteria will be asked to participate the study; all will receive a detailed explanation of the nature of the study, of the aims and objectives. After having signed informed consent, patients will be divided into two groups, based on their clinical status: controlled asthma group (group A) and severe asthma group (group B). Visits will be planned at recruitment, after a run-in period (3-months), meant to optimize first and second lines medical treatment and confirm diagnosis of severe asthma. After the run-in period, patients will be re-evaluated and appropriate medical therapy will be tailored (T0). To group B, therapy with Mepolizumab or Omalizumab will be given, according to standard guidelines. At T0 a blood sample will be collected (3ml) to evaluate eosinophils phenotype by flow cytometry (see below). After 6 months (T1/2) and12 months (T1) patients will be visited again and a blood sample will be taken for flow cytometry analysis.All samples will be properly stored and shipped for analysis, according to best of practice and state of art criteria, within 6 hours from sample's collection. Data will be collected and stored according to the GDPR (General Data Protection Regulation, EU 2016/679). Biological material collected will be used only for the indicated assay and excess material will be discharged.
- Baseline evaluation (T'0) In case of acceptance, and after signing an informed consent, participants will be asked to collect a complete medical history, including allergies and Asthma related comorbidities; a comprehensive physical examination and respiratory function tests consisting of: flow-volume spirometry plus residual volume and DLCO determination. In case of a first diagnosis of asthma a bronchodilator response or a methacholine challenge tests will be obtained. In case of a preexistent diagnosis of asthma, previous examination confirming and proving the diagnosis will be collected.
- Evaluation Tool The following data will be collected at T'0, T'1/2 (six months) and T'1 (twelve months): Clinical assessment: ACT, on demand or rescue therapy, acute exacerbations, medical visits, working absences, hospital admissions. Functional assessment: Flow volume curve, Residual volume, Ig E and eosinophils' count, FeNO, 6 minutes walk test.
- Flow cytometry The investigators will evaluate, by multicolor flow cytometry, the expression of molecules on eosinophils'membrane, using a whole-blood staining protocol, so that blood samples will be minimally manipulated, thus reducing the possibility of artifacts. Briefly, peripheral blood samples will be processed within 6 hours from withdrawal. Samples will be stored at room temperature until processing. Blood samples will be stained, using fluorochrome conjugates antibodies directed towards eosinophils'surface molecules. In particular, the investigators will use antibodies against CD45, CD66, CD15, CD16 and Siglec-8, which will be used to identify eosinophils between other leukocytes; CD63, CD294 (CRTH2, prostaglandin D2 receptor), CD125 (IL-5 receptor), CD193 (receptor for several chemokines, as for example RANTES, Eotaxin, MCP-3, MIP1α), and HLA-DR: these molecules are upregulated with eosinophils activation, so they can have an altered expression in severe asthma patients. After 30 minutes incubation at 4°C, FACS lysing solution (BD Bioscience) will be used to remove erythrocytes, according to manufacturer's instruction. Samples will be resuspended in phosphate buffered saline (PBS) and acquired on a LSRFortessa X-20 (BD Bioscience) flow cytometer, equipped with four lasers.
- Conditions
- Severe Asthma
- Being tested
- anti IL5 receptor antibodies
- Keywords
- severe asthma, eosinophils phenotype, anti-IL5R antibody
- Medical subject headings
- Asthma
What participants receive
What participants receive. In a randomised study each person is assigned to one group by chance.
anti IL5 receptor antibodiesBiological
Subjects with severe uncontrolled asthma will be assigned to treatment with Omalizumab (anti Ig E antibodies) or Mepolizumab (anti IL-5 antibodies) according to medical advice and standardized protocols for asthma treatment.
| Study group | Role | What they receive |
|---|---|---|
| Group A | Experimental | Subject with severe asthma, treated with anti IL5R antibodies Receives: Biological: anti IL5 receptor antibodies |
| Group B | No intervention | Subject with severe asthma, treated with conventional therapy |
What is being measured
The primary outcome is the main question the study is designed to answer.
Measurement by flow cytometry of differences in the percentages of eosinophils expressing activation-related molecules
The presence of activated eosinophils will be evaluated by flow cytometry analysis of the expression of CD193, CD63, CD294, CD125, and HLA-DR on eosinophils membrane.
Results will be expressed in term of percentage (%) of eosinophils expressing the molecules;
Measured: 12 months
Measurement by flow cytometry of the expression levels of activation markers on eosinophils membrane
Activation-associated membrane markers (CD193, CD63, CD294, CD125, and HLA-DR) on eosinophils membrane will be evaluated by flow cytometry and results will be expressed in term of Mean Fluorescence intensity (MFI) of the molecules. MFI is a measure of the amount of the molecule expressed on eosinophils membrane: the higher the MFI, the higher the density of molecules on cellular membrane.
The values will be expressed as arbitrary units (AU), defined by the instrument, comparing the samples with a negative (unstained) control.
Measured: 12 months
Secondary outcomes (2)
Correlation between activated eosinophils phenotype and disease progression
Statistical analysis will be used to define the possible correlation between expression of activation markers on eosinophils (as described in primary outcomes 1 and 2) and the clinical status of the patients, evaluated by Flow volume curve, Residual volume, Ig E and eosinophils' count, 6 minutes walking test.
Measured: 12 months
Change in the percentage of activated eosinophils in the group of patients receiving the biological treatment.
Flow cytometry analysis of eosinophils will be performed before and after one year of treatments with antibodies against IL-5 or IL-5R. Percentages of activated eosinophils will be measured as described in Primary outcome
Measured: 12 months
Timeline
From the start of the study to its completion; the mark is today. Estimated dates are the sponsor’s plan.
- First posted12 Aug 2021
- Study start18 Mar 2021
- Primary completionMar 2022 (estimated)
- Study completionMar 2023 (estimated)
Resources
- Official record on ClinicalTrials.govThe registry entry this page is built from, with its full change history.
- Other trials for Severe AsthmaStudies of the same condition, open and under way.
- What the trial phases meanHow a treatment moves from first tests in people to approval.
More details
For doctors and researchers: how the study is designed, who runs it, and its registry IDs.
Study design
- Study type
- Interventional
- Phase
- Phase 4
- Primary purpose
- Diagnosis
- Allocation
- Randomised
- Intervention model
- Parallel groups
- Masking
- None (open label)
- Enrolment
- 80 participants (estimated)
Sponsor and investigators
- Lead sponsor
- Scarlata, Simone, M.D.
- Sponsor type
- University, hospital or other
- Collaborators
- IRCCS San Raffaele
- Responsible party
- Sponsor
IDs, dates and publications
- ClinicalTrials.gov ID
- NCT05001529
- Sponsor's protocol ID
- EoAG
- Status last verified
- Aug 2021
- First submitted
- 23 Apr 2021
- Last updated
- 12 Aug 2021
- Results posted
- No
- Data monitoring committee
- No
- FDA-regulated drug
- No
- FDA-regulated device
- No
Study at a glance
From the registry record, last updated by the sponsor on 12 Aug 2021. Recruitment status is as the sponsor reported it: confirm with the study team.
- Official title
- Flow Cytometry Analysis of Eosinophils Activity and Membrane Receptors Expression in Severe Asthma Patients: Basal Characterization and Evaluation of Changes Induces by Biological Drugs.
- ClinicalTrials.gov ID
- NCT05001529
- Acronym / sponsor ID
- EoAG
- Conditions
- Severe Asthma
- Keywords
- severe asthma, eosinophils phenotype, anti-IL5R antibody
- Phase
- Phase 4
- Study type
- Interventional
- Recruitment status
- Unknown status
- Enrolment
- 80 participants (estimated)
- Lead sponsor
- Scarlata, Simone, M.D.University, hospital or other
- Collaborators
- IRCCS San Raffaele
- Study start
- 18 Mar 2021 (actual)
- Primary completion
- Mar 2022 (estimated)
- Study completion
- Mar 2023 (estimated)
- First posted
- 12 Aug 2021 (actual)
- Results
- Not posted in the source record
Clinical summary
The research question as the sponsor registered it. This describes what the study plans to test, not what it found.
Asthma is a heterogeneous disease, characterized by reversible airflow obstruction, airway hyperresponsiveness, and airway inflammation, in which 40% of patients exhibit eosinophil-driven pathobiology.The main treatment of asthma is the use of corticosteroid, whose use induces a reduction in eosinophils that is considered a strong predictor of response to treatment. Corticosteroids have remained the mainstay treatment of asthma and reduction in eosinophils has remained the unequivocal predictor of steroid response. The prevalence of asthma, which is expected to increase, it is about 300 million people worldwide. About 5-10% of asthma patients have severe disease, which is defined as asthma that requires high-dose inhaled corticosteroids (ICSs) plus a second controller to prevent it from becoming "uncontrolled" or which remains "uncontrolled" despite this therapy. Patients with severe disease have worse quality of life, and disproportionately high morbidity, mortality, and use of health care resources when compared with their peers with well-controlled disease.The pathophysiology of asthma is complex and heterogeneous between patients, as the disease itself; however, on the basis of immune system involvement, it is possible to define 2 subtypes - or endotypes- of asthma. These endotypes are named T2 (for type 2 cells) high or low, and are defined by the levels of expression of the T2 cytokines, IL-4, IL-5, and IL-13 produced by T helper 2 lymphocytes, and innate lymphoid cell-2.T2 high endotype patients display an increase in the number of blood and sputum eosinophils, and have a better response to the current available biological therapies , such as the administration of mepolizumab (anti IL-5 antibody). Eosinophilic asthma is associated to a more severe clinical phenotype,but patients with a T2 endotype respond better to biological therapies. The hypothesis of the present proposal is that the activation status of these cells, analyzed by the expression of activation markers, can be used to define a new, different, endotype, in which eosinophils, although quantitatively low or normal, are qualitatively more active and aggressive, and could therefore act as an indicator of the progression toward a T2 high endotype.Moreover, the investigators will verify whether a different expression of these molecules on eosinophil's surface might be associated with different clinical response to biologic medications.
Detailed description
Subject satisfying eligibility criteria will be asked to participate the study; all will receive a detailed explanation of the nature of the study, of the aims and objectives. After having signed informed consent, patients will be divided into two groups, based on their clinical status: controlled asthma group (group A) and severe asthma group (group B). Visits will be planned at recruitment, after a run-in period (3-months), meant to optimize first and second lines medical treatment and confirm diagnosis of severe asthma. After the run-in period, patients will be re-evaluated and appropriate medical therapy will be tailored (T0). To group B, therapy with Mepolizumab or Omalizumab will be given, according to standard guidelines. At T0 a blood sample will be collected (3ml) to evaluate eosinophils phenotype by flow cytometry (see below). After 6 months (T1/2) and12 months (T1) patients will be visited again and a blood sample will be taken for flow cytometry analysis.All samples will be properly stored and shipped for analysis, according to best of practice and state of art criteria, within 6 hours from sample's collection. Data will be collected and stored according to the GDPR (General Data Protection Regulation, EU 2016/679). Biological material collected will be used only for the indicated assay and excess material will be discharged.
- Baseline evaluation (T'0) In case of acceptance, and after signing an informed consent, participants will be asked to collect a complete medical history, including allergies and Asthma related comorbidities; a comprehensive physical examination and respiratory function tests consisting of: flow-volume spirometry plus residual volume and DLCO determination. In case of a first diagnosis of asthma a bronchodilator response or a methacholine challenge tests will be obtained. In case of a preexistent diagnosis of asthma, previous examination confirming and proving the diagnosis will be collected.
- Evaluation Tool The following data will be collected at T'0, T'1/2 (six months) and T'1 (twelve months): Clinical assessment: ACT, on demand or rescue therapy, acute exacerbations, medical visits, working absences, hospital admissions. Functional assessment: Flow volume curve, Residual volume, Ig E and eosinophils' count, FeNO, 6 minutes walk test.
- Flow cytometry The investigators will evaluate, by multicolor flow cytometry, the expression of molecules on eosinophils'membrane, using a whole-blood staining protocol, so that blood samples will be minimally manipulated, thus reducing the possibility of artifacts. Briefly, peripheral blood samples will be processed within 6 hours from withdrawal. Samples will be stored at room temperature until processing. Blood samples will be stained, using fluorochrome conjugates antibodies directed towards eosinophils'surface molecules. In particular, the investigators will use antibodies against CD45, CD66, CD15, CD16 and Siglec-8, which will be used to identify eosinophils between other leukocytes; CD63, CD294 (CRTH2, prostaglandin D2 receptor), CD125 (IL-5 receptor), CD193 (receptor for several chemokines, as for example RANTES, Eotaxin, MCP-3, MIP1α), and HLA-DR: these molecules are upregulated with eosinophils activation, so they can have an altered expression in severe asthma patients. After 30 minutes incubation at 4°C, FACS lysing solution (BD Bioscience) will be used to remove erythrocytes, according to manufacturer's instruction. Samples will be resuspended in phosphate buffered saline (PBS) and acquired on a LSRFortessa X-20 (BD Bioscience) flow cytometer, equipped with four lasers.
Study design
- Primary purpose
- Diagnosis
- Allocation
- Randomised
- Intervention model
- Parallel groups
- Masking
- None (open label)
- Number of arms
- 2
| Arm | Type | Receives |
|---|---|---|
| Group A | Experimental | Biological: anti IL5 receptor antibodiesSubject with severe asthma, treated with anti IL5R antibodies |
| Group B | No intervention | –Subject with severe asthma, treated with conventional therapy |
Who may qualify
- Ages
- 18 years and older
- Sex
- All
- Healthy volunteers
- Not accepted
- Age groups
- Adult, Older adult
Key inclusion criteria (2 listed)
- proved diagnosis of severe, refractory eosinophilic asthma, according to GINA recommendations and International ERS/ATS guidelines;
- agreeing to participate this study and signing an informed consent.
Key exclusion criteria (5 listed)
- current smoking habit (both tobacco and e-cigarettes),
- concomitant diseases requiring chronic administration of immunosuppressors, biologic medications or systemic corticosteroids for any disease other than asthma
- History of previous or concomitant acute or chronic disease known to directly or indirectly affect eosinophil count, both in a quantitative and qualitative manner (eosinophilic lung and gastrointestinal diseases, systemic vasculitis, allergic bronchopulmonary aspergillosis, parasitic infections, etc)
- COPD/Asthma overlap.
- Inability or denial to sign the informed consent.
Eligibility is decided by the study team after screening; this overview does not establish that a patient qualifies. Full inclusion and exclusion criteria.
Interventions
| Intervention | Type | Arms |
|---|---|---|
| anti IL5 receptor antibodiesSubjects with severe uncontrolled asthma will be assigned to treatment with Omalizumab (anti Ig E antibodies) or Mepolizumab (anti IL-5 antibodies) according to medical advice and standardized protocols for asthma treatment. | Biological | Group A |
Primary outcome
Measurement by flow cytometry of differences in the percentages of eosinophils expressing activation-related molecules
Time frame: 12 months
The presence of activated eosinophils will be evaluated by flow cytometry analysis of the expression of CD193, CD63, CD294, CD125, and HLA-DR on eosinophils membrane. Results will be expressed in term of percentage (%) of eosinophils expressing the molecules;
Measurement by flow cytometry of the expression levels of activation markers on eosinophils membrane
Time frame: 12 months
Activation-associated membrane markers (CD193, CD63, CD294, CD125, and HLA-DR) on eosinophils membrane will be evaluated by flow cytometry and results will be expressed in term of Mean Fluorescence intensity (MFI) of the molecules. MFI is a measure of the amount of the molecule expressed on eosinophils membrane: the higher the MFI, the higher the density of molecules on cellular membrane. The values will be expressed as arbitrary units (AU), defined by the instrument, comparing the samples with a negative (unstained) control.
2 secondary outcomes
Correlation between activated eosinophils phenotype and disease progression
Time frame: 12 months
Change in the percentage of activated eosinophils in the group of patients receiving the biological treatment.
Time frame: 12 months
Results and safety
Results are not available in the source record.
Research links
- Official record on ClinicalTrials.govLast updated by the sponsor 12 Aug 2021.
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Referral and contact
AzTrials does not enrol participants or take referrals. Contact the study team or a site listed below; they decide who can take part. This study is not recruiting at the moment.
1 site in 1 country: see every site and its status.
Questions about this trial
What is this clinical trial studying?
A Phase 4 study testing anti IL5 receptor antibodies in people with Severe Asthma, run by Scarlata, Simone, M.D. at 1 site in Italy.
The full registered description is in the About this study section and the official record.
Who may be eligible to take part?
The sponsor lists 2 inclusion and 5 exclusion criteria. Eligibility is decided by the study team after screening, so read the full criteria and contact a listed site.
Is this clinical trial currently recruiting?
The record says: status not confirmed, as of the sponsor's last update on 12 Aug 2021. Status can change between updates, so confirm with the study contact before making plans.
Where is the study taking place?
The record lists 1 site in Italy. Whether a site is taking part right now should be confirmed with that site. See every site.
What are the possible benefits and risks?
They depend on the study and the treatment, and no study can promise a benefit. The study team explains the known risks during informed consent, before anyone joins; the record itself does not list them.
What does this phase mean?
Phase 4 studies take place after a treatment is approved, to learn more about its long-term benefits and risks in everyday use.
Not every study follows the same path, and some studies (observational studies, device studies) have no phase.
Are results available for this study?
Results are not available in the source record.
How can a doctor refer a patient or contact the research team?
Contact Simone Scarlata, M.D. (+393939259912, s.scarlata@unicampus.it), or a site near the patient. AzTrials does not enrol participants or take referrals.
What does the primary outcome measure?
Measurement by flow cytometry of differences in the percentages of eosinophils expressing activation-related molecules (time frame: 12 months). The study lists 2 primary outcomes in all. It is what the study was designed to measure, not a finding.
Where can I read the original study information?
Does a listing mean the treatment is approved or proven to work?
No. A listing means the study is registered. It does not show that the treatment is safe, works, or is approved by a regulator.
How often is this information updated?
AzTrials copies changed records from ClinicalTrials.gov every night and reloads the whole registry every week. The sponsor last updated this record on 12 Aug 2021.