Effect of the CYP2C19 Polymorphism in Helicobacter Pylori Eradication
Condition: CYP2C19 Polymorphism; H.Pylori Infection; Therapy
Study type: Interventional
Phase: Phase 4

Summary: 
      Background: Triple therapy efficacy against Helicobacter pylori is low worldwide, thus
      alternatives must be sought to improve eradication. Aim: To determine CYP2C19 genetic
      polymorphism effect on H. pylori eradication.

      Methods: A randomized single blinded clinical trial including 133 patients was carried-out.
      H. pylori infection was confirmed by histology and microbiological test. Antibiotic
      susceptibility to amoxicillin and clarithromycin was performed to avoid confusion bias in
      analysis results. CYP2C19 polymorphism "asterisk" *1, "asterisk"*2 and "asterisk" *3 was
      analyzed by Real time PCR (Roche ®), and nested PCR for CYP2C19 "asterisk" *17 polymorphism.
      Participants were randomized into two groups for different H. pylori therapies, one with
      standard omeprazole doses and another with omeprazole doses depending on CYP2C19
      polymorphism. H. pylori eradicating was verified by stool antigen testing (Meridian ®). The
      general results was analysis by statistical computer program and the effectiveness of each
      therapy was analyzed by intention to treat (ITT) and by protocol (PP).

      The study allowed to know the prevalence of the main polymorphisms of CYP2C19 in
      Bogotá-Colombia, also allowed to know the effectivenesses of the two therapies evaluated for
      H. pylori infection. Additionally, the importance of personalized medicine in H. pylori
      eradication therapy was known.
    

Detailed description: 
      Introduction:

      H. pylori has been declared and ratified as carcinogen I by the World health organization
      (WHO) International agency for research on cancer (IARC). Globally first line therapy
      eradication treatment recommended for H. pylori, combines a proton-pump inhibitor (PPI) with
      two antibiotics (amoxicillin and clarithromycin or metronidazole). However, today
      effectiveness of this therapy is less than 75% "percentage", due mainly to H. pylori
      antibiotic resistance. Other factors involved in therapeutic failure are treatment adherence
      and host genetic factors that may affect medication pharmacokinetics, specifically PPI
      pharmacokinetics PPI play an essential role in eradication therapies, it suppresses deeply
      acid secretion, thus increasing hydrogen ion potential (pH) above 6.0. This allows H. pylori
      to replicate more actively than when stomach pH is less than 6.0, therefore improves
      antibiotic activity promoting greater antimicrobial agent stability and antibiotic
      concentration in stomach. PPIs action depends on its metabolism by cytochrome P450 (CYP)
      enzymes.Recent studies have showed that CYP2C19 genotype can affect PPIs ability to suppress
      acid secretion in the stomach, as has been observed for omeprazole. Omeprazole is metabolized
      by CYP2C19 (90%"percentage") and by CYP3A4 (10%"percentage"). CYP2C19 gene has 21
      polymorphisms, three of which play an important role in PPIs metabolism. Thus, depending on
      polymorphism they are designated as early metabolizers (EM) ("asterisk"*1/"asterisk"*1),
      intermediate metabolizers (IM) ("asterisk"*1/"asterisk"*X) and poor metabolizers (PM)
      ("asterisk"*X/"asterisk"*X). Where "asterisk"*1 allele is the wild type allele ("Wild type")
      and "asterisk"*X correspond to mutated allele. Patients with genotype CYP2C19
      "asterisk"*1/"asterisk"*2 o"asterisk" *1/"asterisk"*3 are IM and those with genotype CYP2C19
      "asterisk"*2/"asterisk"*2 or CYP2C19 "asterisk"*3/"asterisk"*3 are PM. The presence of
      CYP2C19 "asterisk"*2 and CYP2C19 "asterisk"*3 polymorphisms predict the individual
      metabolizer phenotype. In 2006 the CYP2C19"asterisk"*17 allele was found, whose function is
      clinically important because heterozygotes, with CYP2C19"asterisk"*1/"asterisk"*17 forms are
      simply considered ultra-fast metabolizers by other authors. The clinical implication of this
      latter polymorphism relies on the following: if PPIs are quickly metabolized, standard PPIs
      dose fails to adequately suppress acid secretion, and H. pylori eradication therapy will be
      less effective if the microorganism is sensitive to antibiotics. A recent work in Colombia to
      eradicate H. pylori, using triple therapy with clarithromycin or levofloxacin combined with
      amoxicillin, reported that although the organism was sensitive to these antibiotics, there
      was variability with the achieved eradication. These results may suggest the possibility that
      additional factors, different from antimicrobial resistance, can influence the treatment
      effectiveness in our population.

      General Methods:

      The aim of present was to determine CYP2C19 genetic polymorphism influence on H.pylori
      eradication. For this purpose a randomized single blinded clinical trial was done. It was
      conducted from 2012 to 2015 in Bogotá, Colombia. The study was performed in accordance with
      Good Clinical Practice guidelines and ethical principles of the Declaration of Helsinki.
      Ethics Committee of the participating institutions approved the study protocol, where all
      participants signed written informed consent.

      After obtaining informed consent, 356 patients were invited to participate in the study. All
      enrolled participants underwent initial endoscopy, carried-out at the upper endoscopy
      service, clinical center from Bogotá, D.C., Colombia to obtain gastric biopsy for histology,
      microbiological and genotypic test. However, due to the strict inclusion and exclusion
      criteria proposed, of the 356 participants, only 133 were included in the study.

      All participants in the study underwent endoscopy of upper digestive tract under aseptic
      conditions, with a minimum of six hours of fasting. During the endoscopy five biopsies were
      taken from the antrum and four biopsies from the body for histopathology, microbiology and
      molecular analysis. For histopathological analysis, the biopsy samples were sent to pathology
      in separate properly marked containers. For microbiological and molecular analyses three
      antral and three body of the stomach biopsies were collected and used as follows: one biopsy
      from antrum for rapid urease test, two biopsies (antrum and one from the body of the stomach)
      for H. pylori culture and susceptibility test. Culturing was carried out only when one or
      both additional tests were positive for H. pylori. If the culture was negative, but any other
      tests were positive, microorganism detection was confirmed by molecular biology analysis
      (ureA gene detection H. pylori). It was considered a participant was infected with H. pylori
      when two or more tests were positive. Another biopsy was used for DNA extraction and
      subsequent analysis of CYP2C19 genetic polymorphism.

      Culture was performed from biopsies of participants whose rapid urease test and histology
      were positive, from a biopsy of antrum and body of the stomach on Brucella agar (BD) enriched
      with 7% "percentage" v/v horse blood, Isovitalex (BD) and Helicobacter Pylori Selective
      Supplement (Dent) (Oxoid). Following, the samples were incubated at 37°C with 11%
      "percentage"carbon dioxide (CO2) for 3 to 5 days. Antibiotic susceptibility test from pure
      colonies was carried-out by agar dilution (gold standard method) according to the Clinical
      and Laboratory Standards Institute (CLSI) to determine amoxicillin and clarithromycin minimum
      inhibitory concentration (MIC). In addition, CYP2C19 polymorphism
      "asterisk"*1,"asterisk"*2,"asterisk"*3 and "asterisk"*17 were identified for all 133 patients
      included in this study. For this propose DNA was obtained from gastric biopsies by DNA
      Isolation from Human Samples from commercial Kit (QIAGEN), and then the identification of the
      respective polymorphisms was done.

      CYP2C19 "asterisk"*1,"asterisk"*2,"asterisk"*3 polymorphisms were determined by real-time
      polymerase chain reaction (RT-PCR) using Modular Assays kit for human CYP2C19"asterisk"*2 and
      CYP2C19"asterisk"*3 (Roche), according to manufacturer's instructions. And nested PCR and
      Restriction Fragment Length Polymorphism (RFLP) was done for determination of allele
      "asterisk"*17 according with Baldwin et al. report.

      Due to the study compared two types of therapies, the 133 patients were randomized for
      received the treatments into two groups by a computer program. Group one or conventional
      group and group 2 or personalized group. Both groups received amoxicillin and clarithromycin
      but first group received standard doses of omeprazole and in the second group omeprazole
      doses was adjusting according to the CYP2C19 polymorphism of each participant.

      After performing microbiological and molecular testing, the participants were allocated to a
      treatment regimen according to a randomized crossover sequence, provided by a
      computer-generated randomization list. Participants were contacted by telephone for assigned
      an appointment for treatment delivery according to the assigned group.

      All participants were given detailed information about the adverse effects that could occur
      with the different medications. Verification of possible adverse effects was performed
      telephonically using a validated and pre-coded questionnaire that included the following
      symptoms: diarrhea, metallic taste, nausea, bloated feeling, loss of appetite, vomiting,
      abdominal pain, constipation, and rash. The intensity of each symptom was graded from zero to
      three: 0: absent, 3: severe, using a Likert scale. Finally H. pylori eradication was verified
      8 weeks post treatment by antigen test (Meridian®). For this test, each participants was
      requested a stool sample. The test was carried-out according to manufacturer's indications.

      For the analysis of results, a database of the participants in excel and in spss was
      generated. These databases included general characteristics of each participant, result of
      each of the microbiology tests, molecular biology. Descriptive statistics and analysis of the
      effectiveness of the treatments used were carried out.
    

Intervention name(s): amoxicillin clarithromycin omeprazole 1; amoxicillin clarithromycin omeprazole 2
Intervention type(s): Drug; Drug
Intervention description(s): Patients in this received triple standard therapy for eradication of H.pylori with standard doses of proton pump inhibitor (omeprazole), in combination with amoxicillin and clarithromycin in absence of antibiotic resistance.; The treatment for patients in this group was prescribing triple standard therapy for H.pylori eradication with amoxicillin and clarithromycin in absence of antibiotic resistance but with different doses of proton pump inhibitor (omeprazole) according to CYP2C19 genotype in each patients.
Other intervention name(s): Conventional therapy; Group 1; Personalized therapy; Group 2
Intervention MeSH terms: Amoxicillin; Clarithromycin; Omeprazole
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: A randomized single - blinded Clinical Trial conducted from 2012 to 2015 in Bogotá, Colombia. The aim was studying the effectiveness of triple therapy for H. pylori with amoxicillin, clarithromycin and standard dose of omeprazole versus amoxicillin, clarithromycin with dose of omeprazole according to CYP2C19 polymorphisms was studied, the subjects were allocated to a treatment regimen according to a randomized crossover sequence, provided by a computer-generated randomization list. Patients were contacted by telephone for assigned an appointment for treatment delivery according to the assigned group.
Masking: Single (Care Provider)
Primary purpose: Treatment