Healthy VolunteerPhase 4NCT01991990
A Pharmacodynamic Study to Evaluate Neutrophil Distribution Kinetics and Function Following Single-Dose RoActemra/Actemra (Tocilizumab) in Healthy Volunteers
A Phase 4 study testing tocilizumab [RoActemra/Actemra] in people with Healthy Volunteer, run by Hoffmann-La Roche at 1 site in United Kingdom.
Trial summary
This Phase IV, single-blind , randomized, two-arm study will explore the pharmacodynamics effects of RoActemra/Actemra (tocilizumab) on neutrophil redistribution, function and survival in healthy subjects. Read more about this study
- Who is it for?
- Ages 18 to 65 years, men onlyHealthy volunteers can take part
- Conditions the study is for
- Healthy Volunteer
- What is being tested?
- tocilizumab [RoActemra/Actemra]
- Type of treatment
- Injection or infusion
- Could I receive a placebo?
- Yes, some participants may receive a placebo
- How many people
- 18 participants
- Where
- 1 siteUnited Kingdom
- Study dates
- May 2014to Dec 2014
- Phase
- Phase 4
- Are there results?
- Yes, posted in the registrySummary in the doctors' view
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 to 65 years
- Men only
- Healthy volunteers accepted
- Adult, Older adult
Who may be able to join
- Male aged between 18 and 65 years inclusive
- Healthy as determined by screening assessments
- Body mass index (BMI) 18 to 30 kg/m2 inclusive
- Non-smoker
- Must agree to use a barrier method of contraception supplemented with spermicide during the treatment period and for at least 150 days after the last dose of study drug
Who cannot join
- Participation in a clinical study with an investigational drug within 3 months or at least 5 half-lives (whichever is longer) prior to dosing
- Current or past history of smoking within 6 months
- Previous exposure to therapeutic monoclonal antibodies in the past 6 months prior to screening
- Current or clinically significant history of any condition that, in the opinion of the investigator, would: place the subject at undue risk; invalidate the giving of informed consent; interfere with PK or PD data; or interfere with the ability of the subject to complete the study
- History of severe allergic or anaphylactic reactions to humanized or murine monoclonal antibodies
- Any recurrent infections; infection requiring antibiotic treatment in the 6 weeks prior to dosing; mononucleosis in the 6 months prior to dosing; known HIV, Hepatitis B, or Hepatitis C; or active infection at the time of screening
Show all 17Show fewer
- Active tuberculosis (TB) requiring treatment within the previous 3 years.
- Evidence of active malignant disease, malignancies diagnosed within the previous 10 years (except basal cell carcinoma of the skin that has been excised and cured), or breast cancer diagnosed within the previous 20 years
- Primary or secondary immunodeficiency
- Autoimmune disease
- Use or dependence on substance of abuse
- Alcohol abuse or average weekly intake greater than 2 units per day
- Screening or baseline resting heart rate < 45 or >90 beats per minute
- Major surgery within 8 weeks prior to screening
- Major illness in the 3 months prior to dosing
- Biliary obstruction
- Current or past history of diverticulitis
Trial locations and contacts
1 site in 1 country.
About this study
Official title: A Single Blind Phase IV Pharmacodynamic Study to Evaluate Neutrophil Distribution Kinetics and Function Following Single-Dose Tocilizumab Treatment in Healthy Subjects
This Phase IV, single-blind , randomized, two-arm study will explore the pharmacodynamics effects of RoActemra/Actemra (tocilizumab) on neutrophil redistribution, function and survival in healthy subjects. Subjects will receive either a single dose of intravenous (IV) RoActemra/Actemra at a dose of 8 mg/kg over one hour on study Day 0 or placebo. Neutrophil kinetics data will be collected for all subjects up to Day 10 of the study. Following the last study visit on Day 10, all subjects will attend two further safety follow-up visits on Day 28 and Day 56.
- Conditions
- Healthy Volunteer
- Being tested
- placebo, tocilizumab [RoActemra/Actemra]
What participants receive
What participants receive. In a randomised study each person is assigned to one group by chance.
placeboDrug
Single i.v. infusion
tocilizumab [RoActemra/Actemra]Drug
Single 8 mg/kg i.v. infusion
| Study group | Role | What they receive |
|---|---|---|
| Placebo | Placebo comparator | Not described Receives: Drug: placebo |
| RoActemra/Actemra | Experimental | Not described Receives: Drug: tocilizumab [RoActemra/Actemra] |
What is being measured
The primary outcome is the main question the study is designed to answer.
Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir)
On Day 4 participants had neutrophils isolated from 100 milliliters (mL) of acid-citrate dextrose (ACD)-anti-coagulated autologous venous blood and labeled in autologous plasma with up to 2.5 megaBecquerel (MBq) 111 Indium (111In)-tropolonate before being reinjected. Participants rested for 45 minutes (min) post-injection to allow for neutrophil equilibrium between the circulating and marginating neutrophil pools. Whole-body profiling was performed in a heavily shielded dedicated whole-body counter with 2 highly sensitive scintillation detectors with the recorded counts corrected for the physical decay of 111In to allow measurement of the effect of TCZ on the normal redistribution pattern of neutrophils and assessment of margination of neutrophils in the presence of TCZ. Distribution of radiolabelled neutrophils on Day 4 (45 min post re-injection) in the blood, liver/spleen and pelvic bone marrow, expressed as percentages of total body counts (TBCs).
Measured: Day 4
Neutrophil Redistribution Analysis on Day 5
On Day 4 participants had neutrophils isolated from 100 mL of ACD-anti-coagulated autologous venous blood and labeled with up to 2.5 MBq 111In-tropolonate before being reinjected. Participants rested for 45 min post-injection to allow for neutrophil equilibrium between the circulating and marginating neutrophil pools. Whole-body profiling was performed in a heavily shielded dedicated whole-body counter with 2 highly sensitive scintillation detectors with the recorded counts corrected for the physical decay of 111In to allow measurement of the effect of TCZ on the normal redistribution pattern of neutrophils and assessment of margination of neutrophils in the presence of TCZ. Distribution of radiolabelled neutrophils and peak counts, on Day 5 (24-hours post re-injection) in liver/spleen and pelvic bone marrow were decay corrected and expressed as percentages of Day 4 (45 minutes post re-injection).
Measured: Day 5
Neutrophil Redistribution Analysis on Day 10
On Day 4 participants had neutrophils isolated from 100 mL of ACD-anti-coagulated autologous venous blood and labeled with up to 2.5 MBq 111In-tropolonate before being reinjected. Participants rested for 45 min post-injection to allow for neutrophil equilibrium between the circulating and marginating neutrophil pools. Whole-body profiling was performed in a heavily shielded dedicated whole-body counter with 2 highly sensitive scintillation detectors with the recorded counts corrected for the physical decay of 111In to allow measurement of the effect of TCZ on the normal redistribution pattern of neutrophils and assessment of margination of neutrophils in the presence of TCZ. Distribution of radiolabelled neutrophils and peak counts, on Day 10 (6 days post re-injection) in liver/spleen and pelvic bone marrow were decay corrected and expressed as percentages of Day 4 (45 minutes post re-injection).
Measured: Day 10
Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils
Neutrophil phagocytosis was assessed by flow cytometry using heat-killed Staphylococcal pneumonia (S.pneumonia) bacteria labeled with eFluor670. Phagocytosis was quantified by measuring the eFluor670 fluorescence from neutrophils containing phagocytosed bacteria. Experiments were performed using neutrophils (PMN) only, PMN plus S. pneumonia at 4 degrees(˚) centigrade (C) (to control for non-specific bacterial adherence to PMN cell surface), and PMN plus S. pneumonia at 37˚C. Change from baseline in the percentage of eFluor670+ neutrophils was calculated on Day 4.
Measured: Baseline, Day 4
Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils
Neutrophil phagocytosis was assessed by flow cytometry using heat-killed Staphylococcal pneumonia bacteria labeled with eFluor670. Phagocytosis was quantified by measuring the eFluor670 fluorescence from neutrophils containing phagocytosed bacteria. Experiments were performed using neutrophils (PMN) only, PMN plus S. pneumonia at 4˚C (to control for non-specific bacterial adherence to PMN cell surface), and PMN plus S. pneumonia at 37˚C. Change from baseline in the eFluor670+ MFI was calculated on Day 4.
Measured: Baseline, Day 4
Neutrophil Respiratory Burst: Change From Baseline to Nadir (Day 4) in the Production of Reactive Oxygen Species as Measured by Chemiluminescence (Relative Light Units - Absolute)
Neutrophils generate a respiratory burst using reactive oxygen species (ROS) to kill invading pathogens. When luminol is used as a substrate for ROS, a chemical reaction is produced resulting in photon emission (chemiluminescence) in primed and unprimed neutrophils following formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation which is quantifiable. fMLP stimulation of the respiratory burst is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in primed neutrophils. The maximal fMLP response is observed in primed neutrophils and is an ex vivo measure of the capacity of neutrophils to respond to pathogenic stimuli. In the current experiments, neutrophils were primed with tumor necrosis factor alpha (TNFα). Light emission was recorded on a luminometer. Absolute change from baseline in the production of ROS on Day 4 was reported.
Measured: Baseline, Day 4
Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology
Neutrophil apoptosis was measured using microscopy method with slides stained with Diff-Quik (modified Wright Giemsa stain) and morphology examined under oil immersion light microscopy with 100 times magnification. Neutrophils constitutively undergo apoptosis when cultured ex vivo, and this can be delayed by the addition of agents such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or TNFα. Apoptotic neutrophils were characterized with dark and pyknotic nuclei compared to the viable neutrophils. Change from baseline in the percentage of apoptotic neutrophils on Day 4 measured by microscopy is reported.
Measured: Baseline, Day 4
Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry
Ageing neutrophils translocate phosphatidylserine from the inner leaflet of the plasma membrane to the outer leaflet during the early stages of apoptosis. This translocation can be measured due to the affinity of Annexin V (AV) to bind exposed phosphatidylserine. Propidium Iodide (PI) is normally membrane-impermeable but enters cells in late apoptosis when their plasma membrane becomes leaky. Neutrophils constitutively undergo apoptosis when cultured ex vivo, and this can be delayed by the addition of agents such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or TNFα. Apoptosis was assessed by flow cytometry with fluorescein isocyanate-labeled recombinant human AV (AV-FITC) and PI staining and the change from baseline in the percentage of apoptotic neutrophils on Day 4 measured by flow cytometry is reported.
Measured: Baseline, Day 4
Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry
Neutrophil shape change is an indicator of the chemotactic ability of neutrophils to respond to and migrate to sites of inflammation. For determination of neutrophil shape change, fresh (0 min control), phosphate-buffered saline (PBS) control (30 min control) and formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated (30 min fMLP) PMNs (at 5 × 10^6 PMNs/ milliliter [mL]) were fixed with CellFIX (organic solvent used as fixative for adherent cells), 90 microliters (μL) transferred to each sample tube, and cold PBS added to stop further reaction. Shape change was assessed by measuring forward scatter (FSC) on flow cytometry. Change from baseline in the number of neutrophils with shape change on Day 4 was reported.
Measured: Baseline, Day 4
Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Flow Cytometry (FSC-High Cells)
Neutrophil shape change is an indicator of the chemotactic ability of neutrophils to respond to and migrate to sites of inflammation. For determination of neutrophil shape change, fresh (0 min control), PBS control (30 min control) and fMLP-stimulated (30 min fMLP) PMNs (at 5 × 10^6 PMNs/ mL) were fixed with CellFIX, 90 μL transferred to each sample tube, and cold PBS added to stop further reaction. Shape change was assessed by measuring FSC on flow cytometry. Change from baseline in the percentage of neutrophils with shape change on Day 4 was reported.
Measured: Baseline, Day 4
Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology
Neutrophil shape change is an indicator of the chemotactic ability of neutrophils to respond to and migrate to sites of inflammation. For determination of neutrophil shape change, fresh (0 min control), PBS control (30 min control) and fMLP-stimulated (30 min fMLP) PMNs (at 5 × 10^6 PMNs/ mL) were fixed with CellFIX, 90 μL transferred to each sample tube, and cold PBS added to stop further reaction. Shape change was assessed by microscopy with neutrophils classified as shape-changed if they contained > 1 cell surface bleb or irregularity and change from baseline in percentage of neutrophil with shape change on Day 4 was reported.
Measured: Baseline, Day 4
Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules
Neutrophil surface receptor expression may be used to characterize the activation status of neutrophils. Fresh (0 min), PBS control (30 min) and fMLP-stimulated (30 min) PMNs (5 × 10^6 PMNs/mL) were fixed with CellFIX, and 90 μL transferred to each tube containing antibody mixture (2 μL cluster of differentiation [CD] 11b-brilliant violet (BV) 421, 2 μL CD16-FITC, 5 μL CD62L-allophycocyanin (APC) and 5 μL CD162-phycoerythrin [PE]) or isotype control mixture of equivalent volumes. After 30 minutes of incubation on ice and in the dark, cold PBS was added to stop further reaction. Surface marker expressions were quantified by flow cytometry.
Measured: Day 4
Timeline
From the start of the study to its completion; the mark is today. Estimated dates are the sponsor’s plan.
- First posted25 Nov 2013 (estimated)
- Study startMay 2014
- Primary completionDec 2014
- Study completionDec 2014
Resources
- Official record on ClinicalTrials.govThe registry entry this page is built from, with its full change history.
- Posted resultsParticipant flow, outcome measures and adverse events reported by the sponsor.
- Other trials for Healthy VolunteerStudies of the same condition, open and under way.
- More trials run by Hoffmann-La RocheThe sponsor's other registered studies.
- 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
- Allocation
- Randomised
- Intervention model
- Parallel groups
- Masking
- Single blind
- Who is masked
- Participant
- Enrolment
- 18 participants (actual)
Sponsor and investigators
- Lead sponsor
- Hoffmann-La Roche
- Sponsor type
- Industry (pharma, biotech, devices)
- Responsible party
- Sponsor
- Investigators
- Clinical Trials, Study director, Hoffmann-La Roche
IDs, dates and publications
- ClinicalTrials.gov ID
- NCT01991990
- Sponsor's protocol ID
- WA29049
- Status last verified
- Oct 2015
- First submitted
- 18 Nov 2013
- Last updated
- 11 Nov 2015
- Results posted
- Yes, on ClinicalTrials.gov
Study at a glance
From the registry record, last updated by the sponsor on 11 Nov 2015. Recruitment status is as the sponsor reported it: confirm with the study team.
- Official title
- A Single Blind Phase IV Pharmacodynamic Study to Evaluate Neutrophil Distribution Kinetics and Function Following Single-Dose Tocilizumab Treatment in Healthy Subjects
- ClinicalTrials.gov ID
- NCT01991990
- Acronym / sponsor ID
- WA29049
- Conditions
- Healthy Volunteer
- Phase
- Phase 4
- Study type
- Interventional
- Recruitment status
- Completed
- Enrolment
- 18 participants (actual)
- Lead sponsor
- Hoffmann-La RocheIndustry (pharma, biotech, devices)
- Study start
- May 2014
- Primary completion
- Dec 2014 (actual)
- Study completion
- Dec 2014 (actual)
- First posted
- 25 Nov 2013 (estimated)
- Results
- Posted 11 Nov 2015
Clinical summary
The research question as the sponsor registered it. This describes what the study plans to test, not what it found.
This Phase IV, single-blind , randomized, two-arm study will explore the pharmacodynamics effects of RoActemra/Actemra (tocilizumab) on neutrophil redistribution, function and survival in healthy subjects. Subjects will receive either a single dose of intravenous (IV) RoActemra/Actemra at a dose of 8 mg/kg over one hour on study Day 0 or placebo. Neutrophil kinetics data will be collected for all subjects up to Day 10 of the study. Following the last study visit on Day 10, all subjects will attend two further safety follow-up visits on Day 28 and Day 56.
Study design
- Allocation
- Randomised
- Intervention model
- Parallel groups
- Masking
- Single blind (participant)
- Number of arms
- 2
| Arm | Type | Receives |
|---|---|---|
| Placebo | Placebo comparator | Drug: placebo |
| RoActemra/Actemra | Experimental | Drug: tocilizumab [RoActemra/Actemra] |
Who may qualify
- Ages
- 18 to 65 years
- Sex
- Male only
- Healthy volunteers
- Accepted
- Age groups
- Adult, Older adult
Key inclusion criteria (5 listed)
- Male aged between 18 and 65 years inclusive
- Healthy as determined by screening assessments
- Body mass index (BMI) 18 to 30 kg/m2 inclusive
- Non-smoker
- Must agree to use a barrier method of contraception supplemented with spermicide during the treatment period and for at least 150 days after the last dose of study drug
Key exclusion criteria (17 listed)
- Participation in a clinical study with an investigational drug within 3 months or at least 5 half-lives (whichever is longer) prior to dosing
- Current or past history of smoking within 6 months
- Previous exposure to therapeutic monoclonal antibodies in the past 6 months prior to screening
- Current or clinically significant history of any condition that, in the opinion of the investigator, would: place the subject at undue risk; invalidate the giving of informed consent; interfere with PK or PD data; or interfere with the ability of the subject to complete the study
- History of severe allergic or anaphylactic reactions to humanized or murine monoclonal antibodies
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 |
|---|---|---|
| placeboSingle i.v. infusion | Drug | Placebo |
| tocilizumab [RoActemra/Actemra]Single 8 mg/kg i.v. infusion | Drug | RoActemra/Actemra |
Primary outcome
Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir)
Time frame: Day 4
On Day 4 participants had neutrophils isolated from 100 milliliters (mL) of acid-citrate dextrose (ACD)-anti-coagulated autologous venous blood and labeled in autologous plasma with up to 2.5 megaBecquerel (MBq) 111 Indium (111In)-tropolonate before being reinjected. Participants rested for 45 minutes (min) post-injection to allow for neutrophil equilibrium between the circulating and marginating neutrophil pools. Whole-body profiling was performed in a heavily shielded dedicated whole-body counter with 2 highly sensitive scintillation detectors with the recorded counts corrected for the physical decay of 111In to allow measurement of the effect of TCZ on the normal redistribution pattern of neutrophils and assessment of margination of neutrophils in the presence of TCZ. Distribution of radiolabelled neutrophils on Day 4 (45 min post re-injection) in the blood, liver/spleen and pelvic bone marrow, expressed as percentages of total body counts (TBCs).
Neutrophil Redistribution Analysis on Day 5
Time frame: Day 5
On Day 4 participants had neutrophils isolated from 100 mL of ACD-anti-coagulated autologous venous blood and labeled with up to 2.5 MBq 111In-tropolonate before being reinjected. Participants rested for 45 min post-injection to allow for neutrophil equilibrium between the circulating and marginating neutrophil pools. Whole-body profiling was performed in a heavily shielded dedicated whole-body counter with 2 highly sensitive scintillation detectors with the recorded counts corrected for the physical decay of 111In to allow measurement of the effect of TCZ on the normal redistribution pattern of neutrophils and assessment of margination of neutrophils in the presence of TCZ. Distribution of radiolabelled neutrophils and peak counts, on Day 5 (24-hours post re-injection) in liver/spleen and pelvic bone marrow were decay corrected and expressed as percentages of Day 4 (45 minutes post re-injection).
Neutrophil Redistribution Analysis on Day 10
Time frame: Day 10
On Day 4 participants had neutrophils isolated from 100 mL of ACD-anti-coagulated autologous venous blood and labeled with up to 2.5 MBq 111In-tropolonate before being reinjected. Participants rested for 45 min post-injection to allow for neutrophil equilibrium between the circulating and marginating neutrophil pools. Whole-body profiling was performed in a heavily shielded dedicated whole-body counter with 2 highly sensitive scintillation detectors with the recorded counts corrected for the physical decay of 111In to allow measurement of the effect of TCZ on the normal redistribution pattern of neutrophils and assessment of margination of neutrophils in the presence of TCZ. Distribution of radiolabelled neutrophils and peak counts, on Day 10 (6 days post re-injection) in liver/spleen and pelvic bone marrow were decay corrected and expressed as percentages of Day 4 (45 minutes post re-injection).
Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils
Time frame: Baseline, Day 4
Neutrophil phagocytosis was assessed by flow cytometry using heat-killed Staphylococcal pneumonia (S.pneumonia) bacteria labeled with eFluor670. Phagocytosis was quantified by measuring the eFluor670 fluorescence from neutrophils containing phagocytosed bacteria. Experiments were performed using neutrophils (PMN) only, PMN plus S. pneumonia at 4 degrees(˚) centigrade (C) (to control for non-specific bacterial adherence to PMN cell surface), and PMN plus S. pneumonia at 37˚C. Change from baseline in the percentage of eFluor670+ neutrophils was calculated on Day 4.
Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils
Time frame: Baseline, Day 4
Neutrophil phagocytosis was assessed by flow cytometry using heat-killed Staphylococcal pneumonia bacteria labeled with eFluor670. Phagocytosis was quantified by measuring the eFluor670 fluorescence from neutrophils containing phagocytosed bacteria. Experiments were performed using neutrophils (PMN) only, PMN plus S. pneumonia at 4˚C (to control for non-specific bacterial adherence to PMN cell surface), and PMN plus S. pneumonia at 37˚C. Change from baseline in the eFluor670+ MFI was calculated on Day 4.
Neutrophil Respiratory Burst: Change From Baseline to Nadir (Day 4) in the Production of Reactive Oxygen Species as Measured by Chemiluminescence (Relative Light Units - Absolute)
Time frame: Baseline, Day 4
Neutrophils generate a respiratory burst using reactive oxygen species (ROS) to kill invading pathogens. When luminol is used as a substrate for ROS, a chemical reaction is produced resulting in photon emission (chemiluminescence) in primed and unprimed neutrophils following formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation which is quantifiable. fMLP stimulation of the respiratory burst is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in primed neutrophils. The maximal fMLP response is observed in primed neutrophils and is an ex vivo measure of the capacity of neutrophils to respond to pathogenic stimuli. In the current experiments, neutrophils were primed with tumor necrosis factor alpha (TNFα). Light emission was recorded on a luminometer. Absolute change from baseline in the production of ROS on Day 4 was reported.
Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology
Time frame: Baseline, Day 4
Neutrophil apoptosis was measured using microscopy method with slides stained with Diff-Quik (modified Wright Giemsa stain) and morphology examined under oil immersion light microscopy with 100 times magnification. Neutrophils constitutively undergo apoptosis when cultured ex vivo, and this can be delayed by the addition of agents such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or TNFα. Apoptotic neutrophils were characterized with dark and pyknotic nuclei compared to the viable neutrophils. Change from baseline in the percentage of apoptotic neutrophils on Day 4 measured by microscopy is reported.
Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry
Time frame: Baseline, Day 4
Ageing neutrophils translocate phosphatidylserine from the inner leaflet of the plasma membrane to the outer leaflet during the early stages of apoptosis. This translocation can be measured due to the affinity of Annexin V (AV) to bind exposed phosphatidylserine. Propidium Iodide (PI) is normally membrane-impermeable but enters cells in late apoptosis when their plasma membrane becomes leaky. Neutrophils constitutively undergo apoptosis when cultured ex vivo, and this can be delayed by the addition of agents such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or TNFα. Apoptosis was assessed by flow cytometry with fluorescein isocyanate-labeled recombinant human AV (AV-FITC) and PI staining and the change from baseline in the percentage of apoptotic neutrophils on Day 4 measured by flow cytometry is reported.
Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry
Time frame: Baseline, Day 4
Neutrophil shape change is an indicator of the chemotactic ability of neutrophils to respond to and migrate to sites of inflammation. For determination of neutrophil shape change, fresh (0 min control), phosphate-buffered saline (PBS) control (30 min control) and formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated (30 min fMLP) PMNs (at 5 × 10^6 PMNs/ milliliter [mL]) were fixed with CellFIX (organic solvent used as fixative for adherent cells), 90 microliters (μL) transferred to each sample tube, and cold PBS added to stop further reaction. Shape change was assessed by measuring forward scatter (FSC) on flow cytometry. Change from baseline in the number of neutrophils with shape change on Day 4 was reported.
Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Flow Cytometry (FSC-High Cells)
Time frame: Baseline, Day 4
Neutrophil shape change is an indicator of the chemotactic ability of neutrophils to respond to and migrate to sites of inflammation. For determination of neutrophil shape change, fresh (0 min control), PBS control (30 min control) and fMLP-stimulated (30 min fMLP) PMNs (at 5 × 10^6 PMNs/ mL) were fixed with CellFIX, 90 μL transferred to each sample tube, and cold PBS added to stop further reaction. Shape change was assessed by measuring FSC on flow cytometry. Change from baseline in the percentage of neutrophils with shape change on Day 4 was reported.
Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology
Time frame: Baseline, Day 4
Neutrophil shape change is an indicator of the chemotactic ability of neutrophils to respond to and migrate to sites of inflammation. For determination of neutrophil shape change, fresh (0 min control), PBS control (30 min control) and fMLP-stimulated (30 min fMLP) PMNs (at 5 × 10^6 PMNs/ mL) were fixed with CellFIX, 90 μL transferred to each sample tube, and cold PBS added to stop further reaction. Shape change was assessed by microscopy with neutrophils classified as shape-changed if they contained > 1 cell surface bleb or irregularity and change from baseline in percentage of neutrophil with shape change on Day 4 was reported.
Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules
Time frame: Day 4
Neutrophil surface receptor expression may be used to characterize the activation status of neutrophils. Fresh (0 min), PBS control (30 min) and fMLP-stimulated (30 min) PMNs (5 × 10^6 PMNs/mL) were fixed with CellFIX, and 90 μL transferred to each tube containing antibody mixture (2 μL cluster of differentiation [CD] 11b-brilliant violet (BV) 421, 2 μL CD16-FITC, 5 μL CD62L-allophycocyanin (APC) and 5 μL CD162-phycoerythrin [PE]) or isotype control mixture of equivalent volumes. After 30 minutes of incubation on ice and in the dark, cold PBS was added to stop further reaction. Surface marker expressions were quantified by flow cytometry.
Results and safety
Loading the posted results from ClinicalTrials.gov…
Research links
- Official record on ClinicalTrials.govLast updated by the sponsor 11 Nov 2015.
- Posted resultsParticipant flow, baseline, all outcome measures and adverse events.
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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.
Clinical Trials study director
Hoffmann-La Roche
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 tocilizumab [RoActemra/Actemra] in people with Healthy Volunteer, run by Hoffmann-La Roche at 1 site in United Kingdom.
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 5 inclusion and 17 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: finished, as of the sponsor's last update on 11 Nov 2015. 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 United Kingdom. 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. Some participants in this study may receive a placebo. 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?
Yes. Results are posted in the registry (first posted 11 Nov 2015). A summary is in the For doctors view, and the full results are on ClinicalTrials.gov.
How can a doctor refer a patient or contact the research team?
The record lists no contact. The official record names the sponsor, who can be asked directly. AzTrials does not enrol participants or take referrals.
What does the primary outcome measure?
Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) (time frame: Day 4). The study lists 12 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 11 Nov 2015; AzTrials last refreshed its copy on 30 Oct 2026.