A Pharmacodynamic Study to Evaluate Neutrophil Distribution Kinetics and Function Following Single-Dose RoActemra/Actemra (Tocilizumab) in Healthy Volunteers

Study Overview
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Brief Summary
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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.

Official Title
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A Single Blind Phase IV Pharmacodynamic Study to Evaluate Neutrophil Distribution Kinetics and Function Following Single-Dose Tocilizumab Treatment in Healthy Subjects

Conditions
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Healthy Volunteer

Intervention / Treatment
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* Drug: placebo
* Drug: tocilizumab [RoActemra/Actemra]


Participation Criteria
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Eligibility Criteria
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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

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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Male

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Placebo Comparator: Placebo<br> | Drug: placebo<br>* Single i.v. infusion<br>|
| Experimental: RoActemra/Actemra<br> | Drug: tocilizumab [RoActemra/Actemra]<br>* Single 8 mg/kg i.v. infusion<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| 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). | 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). | 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). | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | Day 4 |