Healthy Volunteer
Conditions
Brief 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. 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.
Interventions
DRUGplacebo
Single i.v. infusion
DRUGtocilizumab [RoActemra/Actemra]
Single 8 mg/kg i.v. infusion
Sponsors
Hoffmann-La Roche
Study design
Allocation
RANDOMIZED
Intervention model
PARALLEL
Masking
SINGLE (Subject)
Eligibility
Sex/Gender
MALE
Age
18 Years to 65 Years
Healthy volunteers
Yes
Inclusion criteria
* 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
Exclusion criteria
* 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 * 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
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) | 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 | 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 | 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 | 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 | 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) | 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 | 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 | 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 | 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) | 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 | 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 | 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. |
Countries
United Kingdom
Participant flow
Pre-assignment details
The screening visit was up to 3 weeks before randomization to the first dose of study medication. Out of 23 screened participants; 5 participants discontinued (4=met exclusion criteria; 1=withdrew), 18 participants were included.
Participants by arm
| Arm | Count |
|---|---|
| Placebo Participants received a single dose of placebo-matched to tocilizumab on Day 0. | 6 |
| Tocilizumab Participants received a single dose of IV TCZ at a dose of 8 mg/kg body weight infusion over 1 hour on Day 0. | 12 |
| Total | 18 |
Baseline characteristics
| Characteristic | Placebo | Tocilizumab | Total |
|---|---|---|---|
| Age, Continuous | 29.5 years STANDARD_DEVIATION 11 | 34.5 years STANDARD_DEVIATION 12.6 | 32.7 years STANDARD_DEVIATION 12.01 |
| Sex: Female, Male Female | 0 Participants | 0 Participants | 0 Participants |
| Sex: Female, Male Male | 6 Participants | 12 Participants | 18 Participants |
Adverse events
| Event type | EG000 affected / at risk | EG001 affected / at risk |
|---|---|---|
| deaths Total, all-cause mortality | — / — | — / — |
| other Total, other adverse events | 3 / 6 | 5 / 12 |
| serious Total, serious adverse events | 0 / 6 | 0 / 12 |
Outcome results
Primary
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.
Time frame: Day 4
Population: Safety analysis population
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD62L - 30 min fMLP | 6 median fluoresence intensity | Standard Error 3 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD11b - 30 min fMLP | 39629 median fluoresence intensity | Standard Error 2699 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD162 - 0 min control | 3393 median fluoresence intensity | Standard Error 454 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD162 - 30 min control | 3088 median fluoresence intensity | Standard Error 433 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD162 - 30 min fMLP | 1571 median fluoresence intensity | Standard Error 219 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD16 - 0 min control | 15822 median fluoresence intensity | Standard Error 741 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD62L - 30 min control | 493 median fluoresence intensity | Standard Error 38 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD16 - 30 min control | 13771 median fluoresence intensity | Standard Error 706 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD11b - 30 min control | 13144 median fluoresence intensity | Standard Error 2416 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD16 - 30 min fMLP | 17145 median fluoresence intensity | Standard Error 884 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD62L - 0 min control | 563 median fluoresence intensity | Standard Error 37 |
| Placebo | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD11b - 0 min control | 12633 median fluoresence intensity | Standard Error 2062 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD16 - 0 min control | 16872 median fluoresence intensity | Standard Error 1874 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD62L - 30 min fMLP | 5 median fluoresence intensity | Standard Error 9 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD16 - 30 min fMLP | 18229 median fluoresence intensity | Standard Error 2613 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD11b - 30 min control | 16047 median fluoresence intensity | Standard Error 2058 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD162 - 0 min control | 3253 median fluoresence intensity | Standard Error 895 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD11b - 30 min fMLP | 44481 median fluoresence intensity | Standard Error 5049 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD16 - 30 min control | 14885 median fluoresence intensity | Standard Error 1744 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD162 - 30 min control | 2972 median fluoresence intensity | Standard Error 820 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD11b - 0 min control | 15751 median fluoresence intensity | Standard Error 2043 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD62L - 30 min control | 588 median fluoresence intensity | Standard Error 181 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD162 - 30 min fMLP | 1568 median fluoresence intensity | Standard Error 472 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD62L - 0 min control | 650 median fluoresence intensity | Standard Error 201 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD162 - 30 min fMLP | 1733 median fluoresence intensity | Standard Error 154 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD11b - 0 min control | 17764 median fluoresence intensity | Standard Error 1910 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD11b - 30 min control | 18134 median fluoresence intensity | Standard Error 1836 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD11b - 30 min fMLP | 41339 median fluoresence intensity | Standard Error 2723 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD16 - 0 min control | 15901 median fluoresence intensity | Standard Error 1476 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD16 - 30 min control | 13475 median fluoresence intensity | Standard Error 1269 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD16 - 30 min fMLP | 14707 median fluoresence intensity | Standard Error 2205 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD62L - 30 min control | 615 median fluoresence intensity | Standard Error 37 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD62L - 30 min fMLP | 9 median fluoresence intensity | Standard Error 12 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD162 - 0 min control | 3849 median fluoresence intensity | Standard Error 314 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD162 - 30 min control | 3355 median fluoresence intensity | Standard Error 347 |
| Tocilizumab (PMN-Low Group) | Absolute Median Fluorescence Intensities of Neutrophil Adhesion Molecules | CD62L - 0 min control | 767 median fluoresence intensity | Standard Error 65 |
Primary
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.
Time frame: Baseline, Day 4
Population: Safety analysis population
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry | 30 min control | 4882 neutrophils with shape change | Standard Error 3069 |
| Placebo | Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry | 0 min control | 1613 neutrophils with shape change | Standard Error 842 |
| Placebo | Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry | 30 min fMLP | -1889 neutrophils with shape change | Standard Error 2800 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry | 30 min control | -2309 neutrophils with shape change | Standard Error 4439 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry | 0 min control | -359 neutrophils with shape change | Standard Error 2613 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry | 30 min fMLP | -1667 neutrophils with shape change | Standard Error 4093 |
| Tocilizumab (PMN-Low Group) | Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry | 0 min control | 4790 neutrophils with shape change | Standard Error 1341 |
| Tocilizumab (PMN-Low Group) | Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry | 30 min fMLP | 1524 neutrophils with shape change | Standard Error 3515 |
| Tocilizumab (PMN-Low Group) | Neutrophil Morphology: Change From Baseline to Nadir in the Number of Neutrophils With Shape Change Measured Using Flow Cytometry | 30 min control | 8814 neutrophils with shape change | Standard Error 3016 |
Primary
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.
Time frame: Baseline, Day 4
Population: Safety analysis population
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | 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) | 30 min control | 17.0 percentage of shape changed neutrophils | Standard Error 8 |
| Placebo | 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) | 0 min control | 14.0 percentage of shape changed neutrophils | Standard Error 11 |
| Placebo | 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) | 30 min fMLP | 1.0 percentage of shape changed neutrophils | Standard Error 3 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | 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) | 30 min control | 1.0 percentage of shape changed neutrophils | Standard Error 4 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | 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) | 0 min control | 0.0 percentage of shape changed neutrophils | Standard Error 3 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | 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) | 30 min fMLP | 0.0 percentage of shape changed neutrophils | Standard Error 2 |
| Tocilizumab (PMN-Low Group) | 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) | 0 min control | 2.0 percentage of shape changed neutrophils | Standard Error 2 |
| Tocilizumab (PMN-Low Group) | 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) | 30 min fMLP | -1.0 percentage of shape changed neutrophils | Standard Error 3 |
| Tocilizumab (PMN-Low Group) | 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) | 30 min control | 6.0 percentage of shape changed neutrophils | Standard Error 3 |
Primary
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.
Time frame: Baseline, Day 4
Population: Safety analysis population
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology | 30 min control | 13.0 percentage of shape changed neutrophils | Standard Error 11 |
| Placebo | Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology | 0 min control | 12.0 percentage of shape changed neutrophils | Standard Error 10 |
| Placebo | Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology | 30 min fMLP | 2.0 percentage of shape changed neutrophils | Standard Error 6 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology | 0 min control | -3.0 percentage of shape changed neutrophils | Standard Error 4 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology | 30 min control | -2.0 percentage of shape changed neutrophils | Standard Error 4 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology | 30 min fMLP | 4.0 percentage of shape changed neutrophils | Standard Error 5 |
| Tocilizumab (PMN-Low Group) | Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology | 0 min control | 0.0 percentage of shape changed neutrophils | Standard Error 2 |
| Tocilizumab (PMN-Low Group) | Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology | 30 min fMLP | -1.0 percentage of shape changed neutrophils | Standard Error 3 |
| Tocilizumab (PMN-Low Group) | Neutrophil Morphology: Change From Baseline to the Nadir (Day 4) in the Percentage of Neutrophils With Shape Change Measured by Microscopic Morphology | 30 min control | -1.0 percentage of shape changed neutrophils | Standard Error 1 |
Primary
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.
Time frame: Baseline, Day 4
Population: Safety analysis population
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils | PMN and S.pneumonia at 4˚C | -2 median fluoresence intensity | Standard Error 3 |
| Placebo | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils | PMN only | -1 median fluoresence intensity | Standard Error 3 |
| Placebo | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils | PMN and S.pneumonia at 37˚C | 685 median fluoresence intensity | Standard Error 443 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils | PMN and S.pneumonia at 4˚C | 39 median fluoresence intensity | Standard Error 18 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils | PMN only | 2 median fluoresence intensity | Standard Error 7 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils | PMN and S.pneumonia at 37˚C | 979 median fluoresence intensity | Standard Error 350 |
| Tocilizumab (PMN-Low Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils | PMN only | 2 median fluoresence intensity | Standard Error 2 |
| Tocilizumab (PMN-Low Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils | PMN and S.pneumonia at 37˚C | 810 median fluoresence intensity | Standard Error 217 |
| Tocilizumab (PMN-Low Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in Median Fluorescence Intensity (MFI) of eFluor670+ Neutrophils | PMN and S.pneumonia at 4˚C | 32 median fluoresence intensity | Standard Error 7 |
Primary
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.
Time frame: Baseline, Day 4
Population: Safety analysis population.
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils | PMN and S.pneumonia at 4˚C | -1 percentage of eFlouro+ neutrophils | Standard Error 1 |
| Placebo | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils | PMN only | 0 percentage of eFlouro+ neutrophils | Standard Error 0 |
| Placebo | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils | PMN and S.pneumonia at 37˚C | 4 percentage of eFlouro+ neutrophils | Standard Error 2 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils | PMN and S.pneumonia at 4˚C | 2 percentage of eFlouro+ neutrophils | Standard Error 1 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils | PMN only | 0 percentage of eFlouro+ neutrophils | Standard Error 0 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils | PMN and S.pneumonia at 37˚C | 7.5 percentage of eFlouro+ neutrophils | Standard Error 2 |
| Tocilizumab (PMN-Low Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils | PMN only | 0 percentage of eFlouro+ neutrophils | Standard Error 0 |
| Tocilizumab (PMN-Low Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils | PMN and S.pneumonia at 37˚C | 9 percentage of eFlouro+ neutrophils | Standard Error 2 |
| Tocilizumab (PMN-Low Group) | Neutrophil Phagocytosis: Change From Baseline to Nadir (Day 4) in the Percentage of eFluor670-Positive (eFluoro670+) Neutrophils | PMN and S.pneumonia at 4˚C | 5 percentage of eFlouro+ neutrophils | Standard Error 2 |
Primary
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).
Time frame: Day 10
Population: Safety analysis population. One participant in the PMN-high group was excluded due to external contamination affecting profiling data.
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | Neutrophil Redistribution Analysis on Day 10 | Liver/spleen peak counts (Day 10) | 84.1 percentage of Day 4 counts | Standard Error 6.2 |
| Placebo | Neutrophil Redistribution Analysis on Day 10 | Pelvic peak counts (Day 10) | 180.3 percentage of Day 4 counts | Standard Error 12.9 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Redistribution Analysis on Day 10 | Liver/spleen peak counts (Day 10) | 76.6 percentage of Day 4 counts | Standard Error 3.3 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Redistribution Analysis on Day 10 | Pelvic peak counts (Day 10) | 175.6 percentage of Day 4 counts | Standard Error 14.4 |
| Tocilizumab (PMN-Low Group) | Neutrophil Redistribution Analysis on Day 10 | Liver/spleen peak counts (Day 10) | 96.2 percentage of Day 4 counts | Standard Error 2.9 |
| Tocilizumab (PMN-Low Group) | Neutrophil Redistribution Analysis on Day 10 | Pelvic peak counts (Day 10) | 132.6 percentage of Day 4 counts | Standard Error 5 |
Primary
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).
Time frame: Day 4
Population: Safety analysis population: Includes all the participants who received the single dose of randomized study medication. One participant in the polymorphonuclear leukocyte (PMN)-high group was excluded due to external contamination affecting profiling data.
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) | Liver/Spleen | 50.4 percentage of total body count | Standard Error 1.6 |
| Placebo | Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) | Blood | 26.7 percentage of total body count | Standard Error 4.7 |
| Placebo | Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) | Pelvic marrow | 12.6 percentage of total body count | Standard Error 0.9 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) | Liver/Spleen | 55 percentage of total body count | Standard Error 3.1 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) | Blood | 26.5 percentage of total body count | Standard Error 1.9 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) | Pelvic marrow | 11.7 percentage of total body count | Standard Error 1 |
| Tocilizumab (PMN-Low Group) | Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) | Blood | 30.8 percentage of total body count | Standard Error 5 |
| Tocilizumab (PMN-Low Group) | Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) | Pelvic marrow | 10.8 percentage of total body count | Standard Error 1 |
| Tocilizumab (PMN-Low Group) | Neutrophil Redistribution Analysis on Day 4 (Neutrophil Nadir) | Liver/Spleen | 52.4 percentage of total body count | Standard Error 1.1 |
Primary
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).
Time frame: Day 5
Population: Safety analysis population. One participant in the PMN-high group was excluded due to external contamination affecting profiling data.
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | Neutrophil Redistribution Analysis on Day 5 | Liver/spleen peak counts (Day 5) | 91.0 percentage of Day 4 counts | Standard Error 5.1 |
| Placebo | Neutrophil Redistribution Analysis on Day 5 | Pelvic peak counts (Day 5) | 187.8 percentage of Day 4 counts | Standard Error 14.1 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Redistribution Analysis on Day 5 | Liver/spleen peak counts (Day 5) | 90.3 percentage of Day 4 counts | Standard Error 5 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Redistribution Analysis on Day 5 | Pelvic peak counts (Day 5) | 178.4 percentage of Day 4 counts | Standard Error 18.9 |
| Tocilizumab (PMN-Low Group) | Neutrophil Redistribution Analysis on Day 5 | Liver/spleen peak counts (Day 5) | 105.2 percentage of Day 4 counts | Standard Error 3.3 |
| Tocilizumab (PMN-Low Group) | Neutrophil Redistribution Analysis on Day 5 | Pelvic peak counts (Day 5) | 129.1 percentage of Day 4 counts | Standard Error 7 |
Primary
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.
Time frame: Baseline, Day 4
Population: Safety analysis population
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | 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) | Unprimed neutrophils | -16710 relative light units | Standard Error 12346 |
| Placebo | 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) | TNF α primed neutrophils | 82465 relative light units | Standard Error 16727 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | 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) | Unprimed neutrophils | -5553 relative light units | Standard Error 6409 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | 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) | TNF α primed neutrophils | -45257 relative light units | Standard Error 17540 |
| Tocilizumab (PMN-Low Group) | 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) | Unprimed neutrophils | -2766 relative light units | Standard Error 3291 |
| Tocilizumab (PMN-Low Group) | 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) | TNF α primed neutrophils | 64072 relative light units | Standard Error 16130 |
Primary
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.
Time frame: Baseline, Day 4
Population: Safety analysis population
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology | GM-CSF neutrophils | -1.0 percentage of apoptotic neutrophils | Standard Error 1 |
| Placebo | Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology | Untreated neutrophils | -6.0 percentage of apoptotic neutrophils | Standard Error 3 |
| Placebo | Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology | TNFα neutrophils | -2.0 percentage of apoptotic neutrophils | Standard Error 4 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology | GM-CSF neutrophils | -7.0 percentage of apoptotic neutrophils | Standard Error 5 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology | Untreated neutrophils | -1.8 percentage of apoptotic neutrophils | Standard Error 4 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology | TNFα neutrophils | 3.0 percentage of apoptotic neutrophils | Standard Error 4 |
| Tocilizumab (PMN-Low Group) | Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology | Untreated neutrophils | -6.0 percentage of apoptotic neutrophils | Standard Error 5 |
| Tocilizumab (PMN-Low Group) | Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology | TNFα neutrophils | -4.0 percentage of apoptotic neutrophils | Standard Error 5 |
| Tocilizumab (PMN-Low Group) | Neutrophil Survival: Change From Baseline to the Nadir (Day 4) in the Percentage of Apoptotic Neutrophils as Measured by Microscopic Morphology | GM-CSF neutrophils | -12.0 percentage of apoptotic neutrophils | Standard Error 6 |
Primary
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.
Time frame: Baseline, Day 4
Population: Safety analysis population
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Placebo | Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry | TNFα neutrophils | -3.0 percentage of apoptotic neutrophils | Standard Error 4 |
| Placebo | Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry | Untreated neutrophils | -5.0 percentage of apoptotic neutrophils | Standard Error 2 |
| Placebo | Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry | GM-CSF neutrophils | -4.0 percentage of apoptotic neutrophils | Standard Error 2 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry | GM-CSF neutrophils | -13.0 percentage of apoptotic neutrophils | Standard Error 4 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry | TNFα neutrophils | 3.0 percentage of apoptotic neutrophils | Standard Error 2 |
| Tocilizumab (Polymorphonuclear Leukocyte (PMN)]-High Group) | Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry | Untreated neutrophils | -8.0 percentage of apoptotic neutrophils | Standard Error 4 |
| Tocilizumab (PMN-Low Group) | Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry | Untreated neutrophils | -7.0 percentage of apoptotic neutrophils | Standard Error 5 |
| Tocilizumab (PMN-Low Group) | Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry | TNFα neutrophils | 3.0 percentage of apoptotic neutrophils | Standard Error 5 |
| Tocilizumab (PMN-Low Group) | Neutrophil Survival: Change From Baseline to the Nadir in the Percentage of Apoptotic Neutrophils as Measured by Flow Cytometry | GM-CSF neutrophils | -9.0 percentage of apoptotic neutrophils | Standard Error 8 |