A Study of the Effects of RoActemra/Actemra (Tocilizumab) on Neutrophils in Patients With Active Rheumatoid Arthritis Who Have an Inadequate Response to Biologic and/or Non-biologic DMARDs.

Study Overview
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Brief Summary
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This open-label, single arm study will assess the effect of RoActemra/Actemra (tocilizumab) on neutrophils and monitor safety and benefit-risk of RoActemra/Actemra treatment in patients with active rheumatoid arthritis who have an inadequate response to current biologic or non-biologic disease-modifying antirheumatic drugs (DMARDs). Patients will receive RoActemra/Actemra at a dose of 8 mg/kg intravenously every 4 weeks, either as monotherapy or in combination with their current non-biologic DMARD. Anticipated time on study treatment is 52 weeks.

Official Title
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A 52 Week, Single Center, Open-label Study to Evaluate Neutrophil Function and Survival Effects of Tocilizumab (TCZ) in Patients With Active Rheumatoid Arthritis (RA) on Background Non-biologic DMARDs Who Have an Inadequate Response to Current Non-biologic DMARD and/or Anti-TNF Therapy

Conditions
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Rheumatoid Arthritis

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


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Adult patients, >/= 18 years of age Moderate to severe active rheumatoid arthritis of >/= 6 months duration DAS28 >/= 3.2 at screening and baseline Inadequate response to biologic or non-biologic DMARDs Biologic DMARDs must be withdrawn (approximately 5 half-lives for the agent) before first dose of study drug If continuing on a non-biologic DMARD, dose should be stable for at least 8 weeks Oral corticosteroids must have been at stable dose for at least 25 out of 28 days prior to baseline Exclusion Criteria: Major surgery (including joint surgery) within 8 weeks prior to screening or not recovered from prior surgery Rheumatic autoimmune disease other then RA Functional class IV as defined by the American College of Rheumatology (ACR) classification Prior history of or current inflammatory joint disease other than RA Previous treatment with any cell-depleting therapies Intraarticular or parenteral corticosteroids within 4 weeks prior to baseline Active infection or history of recurrent infection Positive for HIV or hepatitis B or C History of or current primary or secondary immunodeficiency

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Non-Randomized
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Single Arm<br> | Drug: tocilizumab [RoActemra/Actemra]<br>* 8 mg/kg iv every 4 weeks, 52 weeks<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Mean Percentage of Cells Staining Positive for Annexin V Binding in Apoptosis | Aging 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 fluorescein isothiocyanate (FITC)-labeled annexin V to bind exposed phosphatidylserine. Cells that stain positive to Annexin V binding are apoptotic. At 4 hours (hrs) and 20 hrs stimulated and control samples were analyzed for levels of apoptosis. | Visits 2, 3, 5, and 8 (Baseline and Weeks 4, 12 and 24) | 
| Mean Percentage of Cells Staining Positive for Annexin V Binding With Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) | Aging 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 FITC-labeled annexin V to bind exposed phosphatidylserine. Cells that stain positive to Annexin V binding are apoptotic. At 4 hrs and 20 hrs stimulated and control samples were analyzed for levels of apoptosis. GM-CSF is an agent that delays apoptosis. Percentage of cells that stained positive for Annexin V binding in the presence or absence of GM-CSF (GM-CSF delayed or constitutive) were determined by flow cytometry. | Visits 2, 3, 5, and 8 (Baseline and Weeks 4, 12 and 24) | 
| Mean Fluorescence Intensity of CD11b on Neutrophil Surface | Neutrophils were incubated with labeled antibodies against CD11b. Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates with greater adhesion, migration, and ingestion of complement-opsonized particles. | Visits 2, 3, and 5 (Baseline and Weeks 4 and 12) | 
| Mean Fluorescence Intensity of CD18 on Neutrophil Surface | Neutrophils were incubated with labeled antibodies against CD18. Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates with greater adhesion, migration, and ingestion of complement-opsonized particles. | Visits 2, 3, and 5 (Baseline and Weeks 4 and 12) | 
| Mean Fluorescence Intensity of CD62L (L Selectin) on Neutrophil Surface | Neutrophils were incubated with labeled antibody against CD62L (L selectin). Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates with greater adhesion of neutrophils to vessel walls. | Visits 2, 3 and 5 (Baseline and Weeks 4 and 12) | 
| Mean Fluorescence Intensity of CD63 on Neutrophil Surface | Neutrophils were incubated with labeled antibody against CD63b. Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates with greater azurophilic degranulation, an indicator of greater microbe killing. | Visits 2, 3, and 5 (Baseline and Weeks 4 and 12) | 
| Mean Fluorescence Intensity of Interleukin-6 Receptor (Il-6R) on Neutrophil Surface | Neutrophils were incubated with labeled antibody against IL-6R. Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates with greater density of membrane bound IL-6 receptor. | Visits 2, 3, and 5 (Baseline and Weeks 4 and 12) | 
| Mean Fluorescence Intensity of Membrane Bound Tumor Necrosis Factor Alpha (mTNFα) on Neutrophil Surface | Neutrophils were incubated with labeled antibody against mTNF. Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates to a greater density of membrane bound TNF. | Visits 2, 3, and 5 (Baseline and Weeks 4 and 12) | 
| Mean Chemiluminescence (Area Under the Concentration-time Curve [AUC]) of Neutrophil Reactive Species Production Using Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) Stimulation | Using luminol as a substrate for reactive oxidants, a chemical reaction is produced resulting in photon emission (chemiluminescence). fMLP stimulation is mediated through the fMLP receptor on the cell surface. The fMLP response is only observed in primed neutrophils and response is a measure of in vivo priming. Measurements of reactive oxygen species are calculated as total chemiluminescence or the AUC. | Visit 2, 3, 5, and 8 (Baseline and predose at Weeks 4, 12 and 24) | 
| Mean Chemiluminescence (AUC) of Neutrophil Reactive Species Production Using Phorbol 12-Myristate 13-Acetate (PMA) Stimulation | Using luminol as a substrate for reactive oxidants, a chemical reaction is produced resulting in photon emission (chemiluminescence). PMA is a receptor-independent stimulator of the respiratory burst and the PMA response measures the total capacity of neutrophils to generate reactive oxidants. Measurements of reactive oxygen species are calculated as total chemiluminescence or the AUC. | Visit 2, 3, 5, and 8 (Baseline and predose at Weeks 4, 12 and 24) | 
| Percentage of Neutrophils Positive for Propidium Iodide (PI)-Labeled Staphylococcus Aureus (S. Aureus) Uptake | S. aureus were heat killed then labeled with PI and opsonized with AB serum (SAPI). S. aureus was then incubated with the neutrophils for 30 minutes at 37 degrees Celsius. The neutrophils were washed, then the percentage of cells positive for the labeled S. aureus (that is, phagocytosed) was calculated via flow cytometry. A higher percentage represented more active phagocytosis. | Visit 2, 3, 5, and 8 (Baseline and Weeks 4, 12 and 24) | 
| Percentage of Neutrophils Positive for Dihydrorhodamine-123 (DHR) Oxidation | Phagocytosis can be measured by incubating neutrophils with PI-labeled heat killed S. aureus following incubation for 30 minutes. Neutrophils are co-incubated with DHR, which becomes oxidized by the products of the respiratory burst generated during phagocytosis. Fluorescence can then be measured by flow cytometry. | Visit 2, 3, 5, and 8 (Baseline and Weeks 4, 12 and 24) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Disease Activity Score Based on 28-Joint Count (DAS28) | The DAS28 is a combined index for measuring disease activity in rheumatoid arthritis. The index includes swollen and tender joint counts, acute phase response (erythrocyte sedimentation rate [ESR] or C-reactive protein [CRP]), and general health status. The DAS28, which uses a 28 joint count, is derived from the original DAS, which includes a 44 swollen joint count. The DAS28 scale ranges from 0 to 10, where higher scores represent higher disease activity. | Screening, Baseline, and Weeks 4, 8, 12, 16, 20, 24, 36, 48, and 52 | 
| Percentage of Participants With Acceptable and Not Acceptable Benefit-Risk Assessments | Benefit:Risk was defined at the participant level. It was considered acceptable if the DAS28 improvement represented at least a moderate European League Against Rheumatism (EULAR) response. The risks were based on the known adverse event (AE) profile of tocilizumab rather than on the actual AEs experienced by each participant | Weeks 12, 24, and 36 |