Comparative Effects of Azithromycin, Telithromycin and Levofloxacin on Drug Metabolizing Enzymes
Condition: Healthy
Study type: Interventional
Phase: Phase 4

Summary: 
      Studies have previously shown that a broad drug interaction screening can be performed using
      enzyme specific probes such as oral caffeine for CYP1A2, N-acetyltrasferase-2 (NAT-2), and
      xanthine oxidase (XO), warfarin plus vitamin K for CYP2C9, omeprazole for CYP 2C19,
      dextromethorphan for CYP2D6, and midazolam for CYP3A4/5. This combination of probes has been
      validated in the Cooperstown 5+1 Cocktail (5+1).1 The use of the 5+1 cocktail provides
      information on the drug metabolizing enzymes that metabolize 90% of hepatically eliminated
      drugs for a fraction of the costs of the individual studies. Using a cocktail of biomarkers
      reduces the overall cost of drug interaction screening. The purpose of this study is to
      evaluate the effects of three Food and Drug Administration (FDA) approved oral antibiotics
      (azithromycin, telithromycin, and levofloxacin) on metabolism of other medications when taken
      together. This will be determined by the measuring the activity of drug metabolizing enzymes
      following administration of certain drug probes (caffeine, dextromethorphan, omeprazole,
      midazolam, and warfarin with vitamin K). A total of 16 subjects will complete four phases of
      the study: 1) Cooperstown 5+1 alone, 2) Azithromycin plus Cooperstown 5+1, 3) Telithromycin
      plus Cooperstown 5+1, and 4) Levofloxacin plus Cooperstown 5+1.
    

Detailed description: 
      Specific Aims

        1. What is the purpose of this study? What question is it designed to answer?

           To compare the effect of azithromycin, telithromycin, and levofloxacin on activity of 7
           hepatic drug metabolizing enzymes using the Cooperstown 5+1 Cocktail.

           Background

        2. Why is this study important?

      We have previously shown that a broad drug interaction screening can be performed using
      enzyme specific probes such as oral caffeine for CYP1A2, N-acetyltrasferase-2 (NAT-2), and
      xanthine oxidase (XO), warfarin plus vitamin K for CYP2C9, omeprazole for CYP 2C19,
      dextromethorphan for CYP2D6, and midazolam for CYP3A4/5. This combination of probes has been
      validated in the Cooperstown 5+1 Cocktail (5+1).1 The use of the 5+1 cocktail provides
      information on the drug metabolizing enzymes that metabolize 90% of hepatically eliminated
      drugs for a fraction of the costs of the individual studies. Using a cocktail of biomarkers
      reduces the overall cost of drug interaction screening. The FDA has indicated that the use of
      probe drugs can be done in place of specific drug interaction studies.2 This streamlines the
      detection of drug interactions and reduces costs. However, it is important to control for
      genetic polymorphism in drug interaction trials so that genetic makeup does not affect the
      outcome.

      Experimental Design and Methods 3) Describe in detail the experimental design and
      methodology. What information will be collected and how will it be collected? Provide a
      description of the subject's participation from start to finish.

      Prior to any procedure of the study each subject will provide written informed consent. All
      the subjects will be genotyped for CYP2D6, CYP2C9, and CYP2C19 after obtaining informed
      consent for pharmacogenomics. This is to distinguish poor metabolizers from extensive
      metabolizers.

      Prestudy screening will be conducted within 4 weeks of the first study phase. Subjects will
      undergo a complete medical history, social history (including medication, alcohol, and
      tobacco use), physical examination, standard 12-lead electrocardiogram (ECG), and laboratory
      screening to assure that inclusion and exclusion criteria are met. The laboratory screening
      data to be obtained are: complete blood count, prothrombin time (PT), international
      normalized ratio (INR), macroscopic and microscopic urinalysis, electrolytes, blood urea
      nitrogen (BUN), serum creatinine, aspartate transaminase (AST), alanine transaminase (ALT),
      total bilirubin, and serum albumin. Women of childbearing potential (defined as premenopausal
      with no history of hysterectomy or tubal ligation) will undergo a serum pregnancy test during
      the screening visit and a urine pregnancy test prior to each phase of the study. Following
      enrollment, subjects will participate in each of the 4 study phases in a random order.

      Study Design and Procedures This will be a randomized, crossover, open-label study. Prestudy
      screening will be conducted within 4 weeks of the first study phase. Screening visits will
      involve obtaining informed consent, medical history, social history, physical examination,
      ECG, and laboratory tests. Following enrollment, subjects will be randomized (and then
      crossed over) to 4 phases as described in Table 1.

      Table 1. Study Design Study Drugs and Procedure Phase 1 (control) Cooperstown 5+1 Cocktail
      alone. The Cooperstown 5+1 Cocktail consists of the following components:

        1. Caffeine (2 mg/kg orally, rounded to nearest 50 mg) with collection of a 12-hour urine.

        2. Warfarin (10 mg + 10 mg vitamin K orally) with collection of plasma over 96 hours.
           Plasma collection times are at 0, 3, 6, 12, 24, 36, 48, 72 and 96 hours after dosing.
           The INR will be checked at 48 hours. If the INR is >1.7, 5 mg of vitamin K will be
           administered by mouth daily until the INR is 1.2.

        3. Omeprazole (40 mg orally) with collection of a plasma sample 2 hours after dosing.

        4. Dextromethorphan (30 mg orally) with collection of a 12-hour urine.

        5. Midazolam (0.075 mg/kg orally) with collection of 8 plasma samples over 6 hours (only
           for Phase 1) to determine CYP3A activity (MDZ CL/F mL/min). Samples will be drawn
           immediately after midazolam administration (0 minute), then at 5 minute, 0.5, 1, 2, 4, 5
           and 6 hours. Breathing, heart rate, and oxygen saturation will be monitored via pulse
           oximetry for the first 90 minutes after midazolam dose.

           Phase 2 Azithromycin 500 mg qAM for 3 days on an empty stomach. On day 4, the
           Cooperstown 5+1 Cocktail will be administered with a fourth dose of azithromycin given 2
           hours after 5+1 administration.

           Phase 3 Telithromycin 800 mg qAM for 3 days on an empty stomach. On day 4, the
           Cooperstown 5+1 Cocktail will be administered with a fourth dose of telithromycin given
           2 hours after 5+1 administration.

           Phase 4 Levofloxacin 750 mg qAM for 3 days on an empty stomach. On day 4, the
           Cooperstown 5+1 Cocktail will be administered with a fourth dose of levofloxacin given 2
           hours after 5+1 administration.

           During the azithromycin, telithromycin, and levofloxacin phases, plasma samples for
           midazolam will be collected at 0.25, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 12, and 24 hours to
           allow for adequate sampling with drugs that potentially inhibit CYP3A. The 5+1 cocktail
           will be administered on day 4 after a minimum of 8-hour fasting.

           Subjects will not be allowed to eat until 2 hours after administration of study drugs
           (5+1 cocktail or antibiotic).

           Washout periods: One week after each phase except for the azithromycin phase is required
           to ensure the drug is completely eliminated from the body. Three weeks after the
           azithromycin phase is required since azithromycin has a longer half-life.

           Sample Collection and Analysis • For caffeine phenotyping, subjects will collect all of
           their urine for 12 hours after receiving the dose to determine CYP1A2 activity
           [(AFMU+1X+1U)/17U], NAT-2 [AFMU/(AFMU+1X+1U)], and XO [1U/(1X+1U)]. Urine will be
           acidified with ascorbic acid (in vitro) to prevent AFMU conversion.

           • For warfarin phenotyping, blood samples will be obtained at 0, 3, 6, 12, 24, 36, 48,
           72 and 96 hours following warfarin plus vitamin K administration to determine CYP2C9
           activity (S-warfarin CL/F, mL/min). For the first 12 hours, blood will be obtained
           through an intravenous (IV) catheter. Thereafter it will be via venipuncture.

           • For omeprazole phenotyping, one 15 mL blood sample will be drawn 2 hours after the
           oral omeprazole dose to determine CYP2C19 activity (OMP/5OH OMP).

           • For dextromethorphan phenotyping, subjects will collect all of their urine for 12
           hours after receiving the dose to determine CYP2D6 activity (DM/DX).

             -  For midazolam phenotyping, use of oral midazolam gives an assessment of inhibition
                of both gut and hepatic CYP3A.

                  -  For Phase 1 (5+1 cocktail alone), 8 blood samples (10 mL each) will be
                     obtained immediately after midazolam administration (0 minute), then at 5
                     minutes, 0.5, 1, 2, 4, 5 and 6 hours to determine CYP3A activity (MDZ CL/F,
                     mL/min).

                  -  During the azithromycin, telithromycin, and levofloxacin phases, blood samples
                     will be collected at 0.25, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 12, and 24 hours (to
                     allow for adequate sampling with drugs that potentially inhibit CYP3A).

             -  All blood samples (except 15 mL for omeprazole) will be collected in duplicate in
                10 mL EDTA-containing test tubes and centrifuged for 15 minutes at 2800 rpm.

             -  Plasma and urine samples will be frozen at -80C until analysis.

           Data Analysis • Caffeine phenotyping assay: Urine aliquots will be assayed to determine
           urinary concentration of 1U, 1X, 17U, and AFMU. Urine aliquots will be analyzed at
           University of Missouri, Kansas City. Caffeine demethylation ratios will be used to
           express CYP1A2, NAT-2, and XO activity.

           • S- and R-warfarin phenotyping assay: S- and R-warfarin in plasma will be determined
           utilizing high performance liquid chromatography (HPLC) analysis at the University of
           North Carolina, Chapel Hill. Warfarin clearance will be determined using
           non-compartmental analysis. WinNonlin® 3.1 will be used for pharmacokinetic analyses.

           • Omeprazole phenotyping assay: Blood samples will be analyzed at the University of
           North Carolina, Chapel Hill, to determine plasma concentrations of OMP and 5OH OMP. The
           ratio of OMP and 5OH OMP at 2 hours following the oral dose of omeprazole will be used
           to describe CYP2C19 activity.

           • Dextromethorphan phenotyping assay: Urine aliquots will be analyzed at University of
           Missouri, Kansas City, to determine urinary concentrations of DM/DX. The
           dextromethorphan metabolic ratio will be used to describe CYP2D6 activity.

           • Midazolam phenotyping assay: Blood samples will be analyzed to determine plasma
           concentration of midazolam, 1-hydroxymidazolam, and 4-hydroxymidazolam. Analyses will be
           performed at Oneida Research Services, Inc. using a proprietary LC/MS/MS method.
           Midazolam clearance will be used to reflect CYP3A activity and clearance will be
           determined by using non-compartmental analysis of midazolam plasma concentration-time
           data. WinNonlin® 3.1 will be used for pharmacokinetic analyses.

           Statistical Analysis

             -  Sample size: Using =0.05, four treatment phases, and =0.20 (power of 80%), an
                estimated sample size of sixteen volunteers will detect a 25% difference in
                metabolism.

             -  Data will be analyzed using the FDA's standard bioequivalency testing.3 If the drug
                phases fall outside of the 0.8-1.25 range versus the control phase, BE will not be
                shown.

      4) If the research is in part a treatment protocol, identify which parts are routine and
      which parts are being done solely for research.

      This study is for research purposes.
    

Intervention name(s): Cooperstown 5+1 alone; Azithromycin plus Cooperstown 5+1; Telithromycin plus Cooperstown 5+1; Levofloxacin plus Cooperstown 5+1
Intervention type(s): Drug; Drug; Drug; Drug
Intervention MeSH terms: Azithromycin; Levofloxacin; Telithromycin
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: None (Open Label)
Primary purpose: Diagnostic
Keywords: Cooperstown Cocktail; Drug Metabolizing Enzymes; Drug Interactions; Phenotyping