Expression of Pro- and Anti-inflammatory Cytokines During Anti-PCSK9 in Familial Hypercholesterolemia
Condition: Hypercholesterolemia, Familial
MeSH terms: Hyperlipoproteinemia Type II; Hypercholesterolemia
Study type: Interventional
Phase: Phase 4

Summary: 
      Statins have been shown to reduce LDL cholesterol (LCLc) levels, stabilizing atheromatous
      plaque, reversing endothelial dysfunction and decreasing thrombogenesis. Novel
      pharmacological approaches, such as PCSK9 inhibitors (PCSK9i), effectively reduce LDL-c. In
      the clinical setting, there are cases of dyslipidemia showing lack of response to statin,
      known as statin-resistant familial hypercholesterolemia (SR-FH), where patients maintain a
      high cardiovascular risk despite statin therapy. Then, therapeutic alternatives are required.
      PCSK9i has shown to reduce cholesterol levels and risk of cardiovascular disease,
      particularly in patients with statin-resistant familial hypercholesterolemia; and recently,
      it has been hypothesized that PCSK9i have an effect on inflammation. Aim. To evaluate the
      effect of anti-PCSK9 treatment on markers related to the inflammatory response in patients
      with SR-FH.

      Methods. Non-randomized, non-controlled, before-after comparison, quasiexperimental,
      single-center study on patients older than 18 years, with diagnosis statin-resistant FH
      (SR-FH), who were attended at the Cardiology Department, Centro Médico Nacional "20 de
      Noviembre ISSSTE", Mexico City. SR-FH was defined as symptomatic cardiovascular disease
      accompanied by LDL-C concentration higher than 160 mg/dL despite maximally tolerated statin
      dose. Clinical-demographic and anthropometry data were collected during a direct interview.
      Blood sample was processed to obtain glycated hemoglobin complete blood count and serum
      lipids. Likewise, flow cytometry was used to characterize baseline circulating M1-,
      M2-macrophages and monocytes. Multiplexing of plasma samples was used to compare plasma
      fraktaline, IL-1, IL-4, IL-6, IL-8, IL-10, MCP-1 and TNF-alpha. Endpoints consisted of: 1)
      lower serum lipids; 2) modification of pro-inflammatory mediators (neutrophils, lymphocytes,
      NtLR, soluble pro-inflammatory cytokines). Quatitative data were resumed as mean ± SD; while
      categorical data as n(%).One-way T-test was applied. Statistical significance was considered
      if p <0.05.
    

Detailed description: 
      Study Design Non-randomized, non-controlled, before-after comparison, quasiexperimental,
      single-center study.

      Study Population Patients older than 18 years, with diagnosis statin-resistant FH (SR-FH),
      who were attended at the Cardiology Department, Centro Médico Nacional "20 de Noviembre
      ISSSTE", Mexico City. SR-FH was defined as symptomatic cardiovascular disease accompanied by
      LDL-C concentration higher than 160 mg/dL despite maximally tolerated statin dose (Ito MK,
      McGowan MP, Moriarty PM. Management of familial hypercholesterolemias in adult patients:
      recommendations from the National Lipid Association Expert Panel on Familial
      Hypercholesterolemia. J Clin Lipidol. 2011;5(3 Suppl):S38-S45. doi:
      10.1016/j.jacl.2011.04.001.). We excluded patients with infections, neoplasia or under
      oncological therapy. The study was approved by the Research, Biosafety and Ethics Committee
      of the Centro Médico Nacional 20 de Noviembre ID 061.2019.

      Clinical-Demographic characteristics Clinical-demographic characteristics like age, gender,
      past diseases and medication use were collected from a direct interview and verified through
      the medical record. Body mass index (BMI) was calculated as weight/height2 (kg/m2). Blood
      pressure was obtained while the patient was in a seated position and was considered to be the
      mean of three readings taken 5 min apart using a Welch Allyn 767 mobile aneroid
      sphygmomanometer (Welch Allyn Inc.; Skaneateles Falls, NY, USA). Diabetes Mellitus was
      defined according to the guidelines from the American Diabetes Association with one of the
      following conditions (repeated for confirmation at a separate date): 1) Hb A1C ≥ 6.5%; 2)
      fasting glucose ≥ 126 mg/dl; or 3) 2-h plasma glucose ≥ 200 mg/dl during an oral glucose
      tolerance test.

      Complete Blood Count and Lipid Profile Following a 12-h fast, venous blood samples (4 ml)
      were collected into BD Vacutainer® tubes (Becton, Dickinson & Co., Franklin Lakes, NJ, USA)
      containing 1.8 mg ethylenediaminetetra-acetic acid/ml of blood. Standard blood cell count was
      performed immediately. For clinical chemistry blood simple was centrifuged at 1 200 g for 5
      min, at 4°C, separated into fractions and plasma was stored at -80°C prior to analyses. Blood
      cell count and plasma triglycerides, total cholesterol, high-density lipoprotein cholesterol
      (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were determined using routine
      clinical laboratory equipment and a standard auto-analyser (Synchron CX®9 PRO Clinical
      System; Beckman Coulter, Brea, CA, USA). Atherogenic index was calculated as log triglyceride
      / HDL-C.

      Flow Cytometry Blood sample was processed following standardized protocol; then, centrifuged
      to obtain leucocyte population. After three washes, cells were fixed in 4% paraformaldehyde
      and stored for 12 hours. Flow cytometry analysis (MACSQuant Analyzer 10 - Miltenyi Biotec)
      was performed in a total of 1,000,000 events collected for each analysis, and corresponding
      isotype controls were used to set appropriate regions. Cells subpopulations were identified
      by their specific marker combination (CD14+, CD163+ for M1-macrophages; CD14+, CD206+ for
      M2-macrophages; CD16+, CXCR4 receptor and CD16+, CXCR2 for monocytes.

      Plasma Soluble Biomarkers An aliquot of blood sample was immediately centrifuged at 5000rpm/5
      min and plasma was collected. Analytes (fraktaline, IL-1, IL-4, IL-6, IL-8, IL-10, MCP-1 and
      TNF-alpha), using commercially available multiplexing assay (HCYTOMAG-60-08K, MILLIPLEX MAP
      Human Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay Merc, Millipore
      Massachusetts, USA), and following manufacturer instructions; and read in Multiplex reader
      (MAGPIX System, Millipore, Austin, Texas).

      Follow up and Study Endpoints Follow-up was conducted after 6 months of therapy, through
      programmed medical evaluations and blood analyses. Primary endpoint consisted of modification
      in: 1) serum lipids; while secondary end points included: 1) pro-inflammatory mediators
      (neutrophils, lymphocytes, NtLR, soluble pro-inflammatory cytokines); and 2) atherogenic
      index.

      Statistical analysis

      Quatitative data were resumed as mean ± SD; while categorical data as n(%).One-way T-test was
      applied. Statistical significance was considered if p <0.05.
    

Intervention name(s): Evolocumab
Intervention type(s): Drug
Intervention description(s): Evolocumab 140 mg was administered intramuscularly every two weeks at the discretion of their physiciansm according to LDLc response. Clinical-demographic and anthropometry data were collected during a direct interview. Blood sample was processed to obtain glycated hemoglobin complete blood count and serum lipids. Likewise, flow cytometry was used to characterize baseline circulating M1-, M2-macrophages and monocytes. Multiplexing of plasma samples was used to compare plasma fraktaline, IL-1, IL-4, IL-6, IL-8, IL-10, MCP-1 and TNF-alpha. Endpoints consisted of: 1) lower serum lipids; 2) modification of pro-inflammatory mediators (neutrophils, lymphocytes, NtLR, soluble pro-inflammatory cytokines).
Other intervention name(s): repatha
Intervention MeSH terms: Evolocumab
Intervention model: Single Group Assignment
Intervention model description: Non-randomized, non-controlled, before-after comparison, quasiexperimental, single-center study.
Masking: None (Open Label)
Primary purpose: Treatment