The Impact of Glucose Lowering Therapies on Circulating Endothelial Progenitor Cells and Its Mobilising Factor Stromal Derived Factor-1α in Patients With Type 2 Diabetes
Condition: Diabetes Mellitus, Type 2; Cardiovascular Diseases
MeSH terms: Cardiovascular Diseases; Diabetes Mellitus; Diabetes Mellitus, Type 2
Study type: Observational

Summary: 
      This is a cross-sectional observational study aiming to examine and compare the impact of
      incretin based therapies i.e. dipeptidyl peptidase-4 (DPP4) inhibitors and glucagon-like
      peptide-1 (GLP-1) analogues, on endothelial progenitor cells (EPCs) and its mobilising
      factor, stromal derived factor-1 α (SDF-1 α), in patients with type 2 diabetes mellitus
      (T2DM) who are well established on those treatments. EPCs provide vascular protection by
      means of endothelial repair and neogenesis. This endothelial protective effect may
      potentially benefit patients affected by micro or macrovascular complications arising from
      vascular injury e.g. cardiovascular disease in T2DM. The study is of particular interest as a
      small study has shown an increase in level of circulating EPC in patients treated with DPP-4
      inhibitors, thought to be mediated via the up regulation of its mobilising factor SDF-1 α.
    

Detailed description: 
      Diabetes is a chronic condition reported to affect 366 million people globally in 2011 and
      estimated to rise to 552 million by 2030 worldwide, i.e. one in 10 adults. In the UK alone,
      the prevalence is 4.5% and approximately 5 million people are projected to be affected in
      2025. Of the entire population of people with diabetes, Type 2 Diabetes Mellitus (T2DM)
      constitutes more than 90%. In the United Kingdom Prospective Diabetes Study (UKPDS), 50% of
      the people with T2DM presented with micro and/or macrovascular complications at the time of
      diagnosis. It is well established that people with T2DM also have 4 to 5 times greater risk
      of cardiovascular complications compared to the general population, accounting for 80% of
      mortality. Thus, the burden of diabetes and its associated complications on health care
      services are enormous. Prescribing medications for the complications associated with diabetes
      alone costs 3 to 4 times more than the cost of medications for managing diabetes in the
      National Health Service (NHS). Thus, therapies that simultaneously target glycaemic control
      and diabetic complications particularly cardiovascular disease (CVD) independent of their
      glucose lowering effect are desirable.

      The current study aims to evaluate the therapeutic effects of incretin based therapies (DPP-4
      inhibitors versus GLP-1 analogues) and incretin based therapies versus non-incretin based
      treatments on levels of EPCs, SDF-1α and other biomarkers in patients with T2DM established
      on these treatments (receiving for >3 months).

      Study objectives To investigate if treatment with DPP-4 inhibitors in patients with T2DM is
      associated with increased levels of Endothelial Progenitor Cells (EPCs) and Stromal Derived
      Factor-1 α (SDF-1α) compared to those receiving GLP-1 analogues in a cross sectional analysis
      adjusted for confounders.

      Further, the study aims to determine if treatment with incretin based therapies is associated
      with higher circulating levels of EPC and SDF1-α compared to non-incretin based therapies.

      Preparation of Peripheral Blood Mononuclear Cells (PBMCs) for subsequent EPC quantification
      PBMCs will be collected using standard techniques. In brief PBMCs will be isolated from
      venous blood collected into a 9.7ml sodium heparin blood tube (which will subsequently be
      stored at room temperature for up to 4 hours). The blood samples will then be centrifuged
      (1500g for 10 min at 4⁰C) and the plasma layer will be collected for later biomarker analysis
      (aliquots of plasma will be frozen at -80⁰C until use). The buffy coat layer (the white
      opaque layer that lies directly above the red cell pellet) will be used for PBMC isolation.
      To isolate the PBMCs, the buffy coat will be centrifuged at 400g on a Ficoll gradient
      (1.077g/ml) for 30 minutes and the resulting cellular layer will then undergo 3 further
      washes (300g for 10 minutes and 20g for 10 minutes X2) to wash the cells and deplete
      platelets. To calculate the number of PBMC isolated, the pellet will be suspended in a known
      volume of Phosphate Buffered Solution (PBS) (1ml) and a small quantity (10µl) will be
      assessed for quantity and viability under an inverted microscope using a haemocytometer and a
      vital stain (Trypan blue).

      To prevent inter-assay variation due to longitudinal sample collection, the samples will be
      stored in liquid nitrogen vapour (using standard freezing protocols) and quantification of
      the EPCs will be performed as one batch on the stored samples.

      Quantification of EPC EPCs will be quantified as cells showing dual positivity for the cell
      surface / transmembrane markers: Cluster of Differentiation 24 (CD24) and Kinase Domain
      Receptor (KDR). Standardized flow cytometry protocols will be employed. In brief, the cells
      will be thawed and immunofluorescent cell staining will be performed using the fluorescent
      conjugated antibodies: CD34-fluorescein isothiocyanate (FITC) and KDR-phycoerythrin (PE). For
      each patient, a corresponding negative control with IgG2a-FITC-PE will be processed. The
      number of circulating EPCs will be expressed in terms of the total number of cells displaying
      dual positivity for CD24 and KDR per µl blood originally processed.

      Biomarkers SDF-1α analysis and the analysis of biomarkers associated with the prevalence of
      circulating EPCs will be measured using sandwich ELISA and multi-array ELISA technology
      in-line with manufacturer protocols. Analysis of endogenous DPP4 will be assayed using a
      commercial colorimetric assay; again manufacturer protocols will be followed. Biomarker and
      DPP4 analysis will be carried out in one batch on stored sodium heparin plasma samples.

      Statistics Statistical methods and analysis Baseline characteristics will be displayed by
      group (those currently receiving DPP-4 inhibitor, those currently receiving GLP-1analogue,
      and those currently not using DPP-4 inhibitor or GLP-1 analogue) as mean and standard
      deviation for normally distributed continuous variables, median and interquartile range for
      non-normally distributed continuous variables, and counts and percentages for categorical
      variables. The primary outcome (EPC) will be compared by treatment group using linear
      regression, with and without adjustment for potential confounders. The confounders to be
      considered are age, gender, ethnicity, body mass index, duration of T2DM and insulin
      treatment. The mean EPC and its 95% confidence interval in each of the three groups will be
      presented as well as the mean difference between DPP-IV vs GLP-1 treatment group and between
      incretin vs non-incretin treatment groups. A similar analysis will be used for all continuous
      secondary outcomes. Binary outcomes will be compared by groups using logistic regression
      analysis with and without adjustment for the same confounders. All statistical tests will be
      2 sided and p<0.05 will be taken to be statistically significant.

      Sample size Based on the power of 80% and significance at 0.025 (to allow for two primary
      comparisons, i.e. DPP-IV vs GLP-1 users and incretin users vs non-incretin users), 216
      patients (72 in each of the three groups) will be required to detect a difference of 10
      EPC/μl assuming a SD of 19.2 to the power of 24. This sample size will also allow a
      difference of 8 pg/ml of SDF-1α to be detected with 75% power assuming a SD of 16 to the
      power of 24.

      Data management Data collection sheet management All clinical data will be stored in a secure
      area at the Leicester Diabetes Centre. Each enrolled subject will be allocated a unique study
      identification number (ID) so that the electronic database remains anonymous.

      The Data Collection Form (Case report Form, CRF) is the primary data collection instrument.
      Data management check will take place and missing data will be explained where possible. If
      the item is not applicable to the individual case, N/A will be written. All entries will be
      printed legibly in black ink. If any entry error has been made, to correct the error, a
      single line will be drawn through the incorrect entry and the correct data entered above it.
      All such changes will be initialled and dated. For clarification of illegible or uncertain
      entries, the clarification will be printed above the item and this will be initialled and
      dated.

      Documentation storage, access, security, archiving All study documentation containing
      identifiable patient data will be managed in accordance with International Conference on
      Harmonisation-Good Clinical Practice (ICH-GCP), Research Governance Framework for Health and
      Social Care and the Data Protection Act. Information will only be obtained from the patient
      if necessary for the study.

      All electronic data will be stored on secure university (University of Leicester) or hospital
      (University Hospitals of Leicester NHS Trust) network drives, to which only the relevant
      study staff have access, which is granted by the research team.

      All study documents and data will be kept for 5 years or the minimum determined by the
      regulatory authorities, whichever is longer. The study file will be archived in line with the
      Trust policy.

      Data confidentiality Each participant will be assigned a unique identification number upon
      recruitment. Patients' contact details will be held on a separate database and used to
      arrange study visits. The database will be password protected and only members of the
      research team contacting patients will have access. All data collected during the study will
      be stored anonymously on a separate database. Again access will be password protected and
      restricted to relevant members of the research team. Electronic data will be stored in the
      University or NHS system.

      Safety Issues The Investigators do not foresee any adverse events over and above those
      associated with everyday life and routine health care that could be attributable to the
      study. The study involves only one venepuncture to withdraw venous blood and hence carries a
      very low risk of having untoward effects. However, all participants will undergo venepuncture
      which can occasionally result in bruising, swelling and temporary discomfort.

      The Investigators will follow the University of Leicester guidelines for managing and
      reporting a Serious Adverse Event (SAE) or Suspected Unexpected Serious Adverse Reaction
      (SUSAR), which follow those outlined in Good Clinical Practice (GCP) guidance.

      A SAE is any adverse event or unexpected adverse reaction that results in death is
      life-threatening requires hospitalisation or prolongation of existing hospitalisation results
      in persistent or significant disability or incapacity consists of a congenital anomaly or
      birth defect. In addition, the Investigators will also define an event as serious if it is an
      important and significant medical event that may not be immediately life threatening or
      resulting in death or hospitalisation but, based upon appropriate medical judgement may
      jeopardise the patient or may require intervention to prevent one or more outcomes listed
      above. Adverse events which do not fall into these categories are defined as non-serious.

      All SAEs will be reported internally to the University Hospitals of Leicester (UHL) NHS Trust
      Research & Development (R&D) and the sponsor (University of Leicester) using appropriate
      reporting forms, within 24 hours of the study team becoming aware of the event. The principal
      investigator is responsible for the review and submission of any SAE, or in their absence,
      another member of the team (in order to avoid a delay). The investigator site file will
      contain documentation for SAE reports and evidence of timely submissions.
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        Male and female patients aged between 35 and 70 years of age (inclusve) who have
        established Type 2 Diabetes mellitus (T2DM) and who have been receiving either (a) DPP-4
        inhibitors, (b) GLP-1 analogues, or (c) non-incretin based treatments for at least 3 months
        (or more).
      
Biospecimen description: 
      Peripheral Blood Mononuclear Cells (PBMCs) for subsequent EPC quantification:

      PBMCs will be isolated from venous blood collected into a 9.7ml sodium heparin blood tube.
      EPCs will be quantified as cells showing dual positivity for the cell surface / transmembrane
      markers: CD24 and KDR (Kinase Insert Domain Receptor). SDF-1α analysis and the analysis of
      biomarkers associated with the prevalence of circulating EPCs will be measured using sandwich
      ELISA and multi-array ELISA technology in-line with manufacturer protocols. Analysis of
      endogenous DPP4 will be assayed using a commercial colorimetric assay; again manufacturer
      protocols will be followed. Biomarker and DPP4 analysis will be carried out in one batch on
      stored sodium heparin plasma samples.