Effect of Stomach Reduction Surgery on the Parameters of Amoxicillin
Condition: Bariatric Surgery Candidate
Study type: Interventional

Summary: 
      The objective of this study was to evaluate the impact of bariatric surgery on the
      pharmacokinetics of amoxicillin.

      Methods: This study was performed as a randomized, open-label, single-dose clinical trial,
      with two periods of treatment, in which obese subjects (n=8) received amoxicillin 500 mg
      capsule orally before and 2 months after the Roux-en-Y gastric bypass surgery. The
      amoxicillin plasma concentration was determined by liquid chromatography coupled to mass
      spectrometry (LC-MS/MS).
    

Detailed description: 
      Study population

      Eight obese volunteers of both sexes, aged between 18 and 60 years old, were selected from
      the obesity program of Hospital General Dr. César Cals with indication of bariatric surgery
      based on the criteria established by the Federal Medical Council (2015) and who were operated
      using the RYGB surgery technique.

      All subjects signed the informed consent form. The study was approved by the institutional
      review board (IRB) of the Federal University of Ceará, accredited by the National Commission
      for Research Ethics - National Health Council, Ministry of Health and the Scientific
      Committee of the Hospital General Dr. César Cals. The study was performed according to the
      ethical principles of the Declaration of Helsinki as revised in 2013 and Good Clinical
      Practice Guidelines.

      Study design and pharmacokinetic sampling

      This study was performed as a monocentric, open-label, randomized, single-dose study with two
      treatment periods - before the surgery (Time 0 - T0) and 2 months after the bariatric surgery
      (Time 2 - T2). In each period, the obese volunteers received a single oral dose of
      amoxicillin 500 mg capsule (Amoxil®, GlaxoSmithKline Brazil Ltda.) with 200 mL water after an
      overnight fast (approximately 8 h). The subjects remained fasted for up to 3 h after taking
      the medication. Water and food restriction was held up to 3 h after the intake of the
      formulation. Standard meals were offered to all subjects. The volunteers who needed to use
      any medication (antihypertensive, antidiabetic and others) were instructed to use it 3 h
      after the intake of amoxicillin. Blood samples (6 mL) for determination of plasma
      concentration of amoxicillin were collected via a venous catheter into heparinized tubes at
      pre-dose (0 h), 0.33, 0.66, 1, 1.5, 2, 2.5, 3, 4, 6 and 8 h post-dose. The plasma samples for
      chromatographic analysis were obtained by centrifugation (2000 g x 12 min at 4 ºC) of the
      blood samples. Plasma samples were stored at -20 °C until the analysis.

      Anthropometric parameters before and after surgery

      At each visit (T0 and T2), the weight and height of all volunteers were measured, followed by
      the calculation of their body mass index (BMI) for anthropometric comparison before and after
      bariatric surgery. The body weight was measured using a digital scale, and the volunteers
      were wearing light clothes, barefoot, with empty pockets and without accessories. The height
      was measured using a stadiometer with 2 meters extension. The subjects were barefoot, with
      legs and feet parallel, weight distributed on both feet, arms relaxed at the side of the body
      and palms facing the body and head positioned on the Frankfurt plane.

      Bariatric surgery procedure

      All volunteers included in this trial were operated using the RYGB surgery technique,
      performed by the same surgical team, following a standardization for the alimentary loop size
      (1.20 m length) and the biliopancreatic loop (1 m length). The gastrojejunoanastomosis was
      calibrated by a Fouchet 32F probe, resulting in a diameter of 12 mm.

      Analytical method

      Amoxicillin plasma concentration was quantified by liquid chromatography coupled to mass
      spectrometry (LC-MS/MS) and a liquid-liquid extraction.

      To sample preparation, 100 µL of each plasma sample, 50 µL of the internal standard (IS)
      cephalexin (2.5 µg/mL) and 500 µL of acetonitrile (100 %) were added into a glass tube. The
      samples were vortexed (30 sec) and centrifuged (2000 g x 4 min at 4 ºC). The organic phase
      was transferred to another glass tube, which was added of 500 µL of deionized water and
      vortexed (10 sec). The samples were added of 10 mL of dichloromethane (100 %), vortexed (10
      sec) and centrifuged (2000 g x 4 min at 4 ºC). The organic phase was transferred to the
      auto-injector PCR plates.

      The high pressure liquid chromatography (HPLC) system (Agilent, Germany) consisted of a
      LC-10AD pump, autoinjector model CTC HST PAL/ 110695 and a G1316A/DE03018295 oven. The
      extracted solutions (10 µL) was injected into a 150 x 4.6 mm (5 μm particles) Inertsil
      ODS-C18 column maintained at 65 ◦C and the mobile phase was methanol/water (30/70, v/v) + 0.1
      % formic acid at a flow rate of 900 μL/min with a split ratio of 1:3. The mass spectrometer
      used was a Quattro Micro (Micromass, Manchester, UK) with electrospray in positive mode for
      MRM (Multiple Reaction Monitoring) to monitor the transitions 366.00 >348.90 and 348.00
      >158.00 for amoxicillin and cephalexin, respectively.

      The method validation was performed according to the United States Food and Drug
      Administration (FDA) bioanalytical method validation guidance [22] and the Brazilian National
      Sanitary Surveillance Agency (ANVISA) [23]. To evaluate the specificity of the method, eight
      different blank plasma lots (4 normal, 2 haemolysed and 2 lipemic) were tested for its
      interferences using the proposed extraction procedure and the chromatographic or
      spectroscopic conditions and compared with those obtained in the samples processed from the
      lower limit of quantification (LLOQ). The calibration curves were prepared by assaying
      standard plasma samples at eight concentrations of amoxicillin (20-5000 ng/mL) in triplicate
      and the linearity of each calibration curve was determined by plotting the peak area ratio
      (y) of amoxicillin/IS vs. nominal concentration of analyte. The calibration curve was
      constructed by weighted (1/x) least squares linear regression. The accuracy and precision of
      assay were evaluated by intra- and inter-assay studies. Seven aliquots of LLOQ and each
      quality control (QC) plasma samples (60, 850, 2500, 4000 and 12000 ng/mL) were run in three
      validation batches on three different days. Inter and intra-day precisions were determined as
      coefficient of variation, CV (%) = (SD/M) x 100 and the accuracy as the percentage relative
      error, RE (%) = [(E - T)/T] x 100, where M is the mean, SD is the standard deviation of M, E
      is the experimentally determined concentration and T is the theoretical concentration. The
      matrix effect experiments were performed using the ratio between amoxicillin (60 and 4000
      ng/mL) and IS injected directly into the mobile phase and standard solutions added to blank
      plasma extracts (4 normal, 2 haemolysed and 2 lipemic). Each sample was obtained by a matrix
      factor normalized by IS (MFN) according to the following formula: MFN = (response of the
      analyte in matrix/internal standard response matrix)/(response of the analyte in
      solution/response of the internal standard solution). Stability QC plasma samples (60 and
      4000 ng/mL) were subjected to short-term (6.0 h) room temperature, three freezes-thaw (-20 to
      25 °C) cycles and 46.5 h autosampler (25 ºC) stability tests in triplicate. The stability
      results were compared with the nominal values.

      Pharmacokinetic analysis

      The pharmacokinetic parameters were obtained based on the plasma concentration vs. time
      curves analyzed by a non-compartmental model using the Phoenix WinNonlin® software, version
      5.0 (Pharsight Corp, MoutainView, CA, USA). Values for the maximum plasma concentration
      (Cmax) and time to Cmax (tmax) were obtained directly from the observed individual plasma
      concentration vs. time curves. The area under the plasma concentration vs. time curve from
      time zero to the time of the last quantifiable concentration was calculated by the linear
      trapezoidal rule. Elimination rate constant (ke) was obtained by log-linear regression
      analysis, and the elimination half-life (t1/2) was calculated with the equation
      [t1/2=ln(2)/ke].

      Statistical analysis

      The quantitative variables (continuous and discrete) were initially analyzed by the
      Kolmogorov-Smirnov test to verify the normality of the distribution. As this requirement was
      verified in most cases, mean and standard deviation (SD) were calculated for descriptive
      statistics. Parametric tests were used for analytical statistics.

      To compare the pharmacokinetic parameters of the obese volunteers obtained before (T0) and
      after (T2) bariatric surgery, the t-test for paired data was used. The mean difference (or
      mean of differences) and their respective 95% confidence interval (95% CI) were also
      determined. Furthermore, the ratio between the parameter values verified before and after
      bariatric surgery was calculated, being expressed as geometric mean and its 95% CI. The
      degree and sign of the linear correlation between two variables were evaluated by the Pearson
      correlation coefficient, accompanied by its 95% CI.

      In all analyses, two-tailed tests were used, establishing the level of significance at 0.05.
      The GraphPad Prism software, version 5.00 (GraphPad Software, San Diego, California, USA) was
      used for both statistical analyses and graphics.
    

Intervention name(s): Amoxil 500 MG Oral Capsule
Intervention type(s): Drug
Intervention MeSH terms: Amoxicillin
Intervention model: Single Group Assignment
Intervention model description: Monocentric, open-label, single-dose study with two treatment periods - before the surgery (Time 0 - T0) and 2 months after the bariatric surgery (Time 2 - T2).
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: Bariatric surgery; Pharmacokinetics; Amoxicillin; Obesity