Coffee Effect in HCV-related Hepatitis
Condition: Chronic HCV-related Hepatitis
MeSH terms: Hepatitis A; Hepatitis
Study type: Interventional
Phase: Phase 3

Summary: 
      Background: In patients with chronic HCV-related liver damage, coffee is associated with a
      reduced risk of progression and of hepatocellular carcinoma (HCC) development. Aim: This
      prospective trial is aimed at assessing the mechanisms underlying the protective effect of
      coffee on evolution in cirrhosis and HCC. Trial design/methods: Forty patients with
      HCV-related hepatitis will be recruited and randomized into two groups: the first will
      consume 4 coffee cups/day/1 month, while the second will remain coffee "abstinent". At day
      30, the two groups will be switched over and exposed to coffee or not for a second month.
      Before entering the study (time 0), during coffee exposure and during abstinence we will
      evaluate the following parameters: liver function tests, viral load, 8-hydroxydeoxyguanosine
      (a marker of oxidative DNA damage), telomere length, apoptosis and collagen deposition.
    

Detailed description: 
      Patients Forty patients with HCV-related chronic liver disease referring to the
      Gastroenterology Unit of Padua out-patient clinic will be prospectively recruited and will
      enter the study.

      In all patients the presence of anti-HCV antibodies will be assayed by a third-generation
      immunoenzymatic test (Ortho Diagnostic Systems, Raritan, NJ) and by confirmatory test
      (recombinant immunoblotting assay, Chiron Corp., Emeryville, CA). A standardized genotyping
      assay (Inno-Lipa HCV III, Innogenetics, Gent, Belgium) will be used.

      Only patients who fulfill the following inclusion criteria will be recruited:

        -  HCV-related chronic hepatitis or cirrhosis, with bioptic (within the previous 24 months)
           confirmation or a clinical diagnosis in case of cirrhosis; (Prothrombin Time - PT, White
           Blood Cells - WBC and platelets - PLT, Ultra Sound - US examination);

        -  anti-HCV and HCV-RNA positivity with Aspartate aminotransferase (AST)/Alanine
           aminotransferase (ALT) at least 1.5x;

        -  age range 30-80 years;

        -  no ongoing interferon treatment, previous treatment with no response or relapse was
           accepted.

      The study is approved by the Hospital Ethical Committee (Prot. 1755 P, Azienda Ospedaliera di
      Padova), the patients' participation is voluntary and each patient signs an informed consent
      to participation, an information note being delivered to each patient's attending general
      practitioner.

      Before the beginning of the intervention phase, for each patient, demographical data will be
      collected, together with information on coffee and smoking habits, alcohol, fruit, vegetable,
      anti-inflammatory drugs consumption, energy and nutrients intake, as potential bias. All
      these data will be assessed by using a quantity-frequency questionnaire designed for use in
      the present study. At time 0 (T0) the patients will be categorized into two groups in
      relation to their routine coffee intake: 0-2 cups/day and 3-5 cups/day. This cut-off was
      chosen because in most papers an effect of coffee consumption is observed for over 2
      cups/day.

      Subjects will be asked to maintain, throughout the study, their alimentary and voluptuary
      habits, while the use of other caffeine-containing beverages is forbidden.

      Trial design of the study

      Patients will be investigated for all the markers at the beginning of the study (T0) and then
      randomized using a computer-generated list (StatsDirect statistic program) in two groups,
      with either 4 coffee cups/day intake for 1 month or abstinence for the same time period.
      Obviously, blinding is not possible and we decided not to prescribe any wash-out period,
      being the T0 sampling examined in relation to the patients' routine coffee consumption.

      After 30 days, the patients will be re-tested, and then shifted over to the opposite
      "treatment" (into the coffee arm those who were abstinent in the first month and vice versa
      those who consumed coffee) and again checked after the second time period.

      Each patient is therefore tested three times, at T0, when he/she enters the study, after
      coffee and following abstinence, being each patient the control of himself in the two phases.
      For seek of simplicity all data obtained following coffee consumption will be compared with
      all the data obtained during abstinence, irrespective of the time sequence of
      exposure/abstinence.

      Considering (on the basis of published data) an expected coffee effect in at least 40% of
      exposed subjects, with 1 control for case, with 5% alpha error and with 80% power, the sample
      size required (20 cases and 20 controls) corresponded to the selected sample for the study.
      Additionally, in this study, given the cross over design in which each patient is
      investigated twice, once during coffee exposure and once during abstinence, the actual number
      of patients investigated is of 40 per arm, twice as high therefore, than required.

      The patients will be given a form in which they registered any deviation and compliance with
      respect to coffee intake and appropriate telephone contacts to report immediately any side
      effect. To reduce the risk of any bias due to inter-individual changes in the preparation of
      coffee, the patients will be provided with typical Italian-style coffee machine of the
      stove-top type (MokaTMBialetti), the required amount of coffee for the preparation of the 4
      coffee cups/day (8 g for each coffee preparation) and instructions on how to prepare it.

      The coffee brand is a 100% Coffea arabica product from the shelf. We also want to analyze the
      product we use. The analyses will be performed by a contract lab (Eurofins analytic GmbH -
      Hamburg-Germany).

      The patients are instructed not to drink any differently prepared coffee and had to report in
      an appropriate form any deviation from the study.

      Methods Blood samples will be collected and stored at -20°C for no longer than 3 weeks.

      In this study the following parameters will be evaluated:

        1. AST/ALT, gammaglutamyl-transpeptidase (γGT) and alkaline phosphatase (ALP) levels;

        2. viral load (HCV-RNA titer),

        3. markers of oxidative damage: 8-hydroxydeoxyguanosine (8-OHdG), nitric oxide (NO),
           Advanced Oxidation Protein Products (AOPP),

        4. marker of genomic stability: Telomere Length (TL),

        5. markers of cell death: M30 and M30/M65,

        6. markers of liver fibrosis and angiogenesis: Procollagen Type III (PIIINP) and Vascular
           Endothelial Growth Factor (VEGF).

      Liver Function Tests determination Serum levels of AST and ALT, gammaGT and ALP will be
      determined as part of the routine clinical procedure.

      HCV-RNA determination The presence of HCV-RNA in the serum will be assessed by amplification
      with PCR of the 5' untranslated region (5'-UTR) of HCV, the most conserved region of the
      virus. The test is performed in all subjects positive for anti-HCV antibodies (screening
      test). The amplification will be performed in two steps, by 'nested PCR'.

      8-OHdG determination This assay will be carried out as follows: in brief, this assay consists
      of 3 steps: (i) genomic DNA extraction using a Wizard Genomic DNA Purification Kit (Promega
      Italia, Milano); (ii) nuclease P1 and alkaline phosphatase hydrolysis of DNA; (iii) 8-OHdG
      determination using an HPLC equipped with an electrochemical detector (HPLC-ED) (ESA
      Coulochem II 5200 A, Bedford, MA). The 8-OHdG levels are expressed as the number of 8-OHdG
      adduct/105 dG.

      NO determination Serum NO levels will be spectrophotometrically assayed by measuring the
      accumulation of its stable degradation products, nitrate and nitrite (Oxford Biomedical
      Research, USA). NO levels will be expressed as µmol/L, as a marker of nitric oxide synthase
      activation and nitrosation.

      AOPP determination Plasma AOPP levels will be measured by spectrophotometric method performed
      by Witko-Sarsat. AOPP concentrations are expressed as µmol/L of chloramines-T equivalents.

      TL analysis by quantitative PCR Genomic DNA extracted will be used also to measure TL by
      using method developed by Cawthon. Two 96-well plates were prepared for each experiment, one
      containing telomere primers and the other for 36B4, encoding acidic ribosomal phosphoprotein
      P0 used as the single copy gene .

      Each 25 µl PCR reaction included 40 ng of DNA, SYBR Green Master Mix (Applied Biosystems,
      Foster City, CA) and primers at final concentrations 270 nM Tel-1 and 900 nM Tel-2 or 300 nM
      36B4u and 500 nM 36B4d respectively.

      PCR amplification is performed in a ABI PRISM 7900 (Applied Biosystem). In each plate, a
      standard curve is produced ranging from 4 to 80 ng of human reference DNA (Applied
      Biosystem). The thermal cycling profile for the telomere amplification is 95°C for 10 min,
      followed by 30 cycles of 95°C for 5 s, 56°C for 10 s and 72°C for 60 s or 30 cycles of 95°C
      for 5 s, 58°C for 10 s and 72°C for 40 s. Following amplification, a dissociation curve is
      performed in order to confirm the specificity of the reaction. All samples are assayed in
      triplicate to test the reproducibility of assay and in each assay a negative control is
      evaluated.

      Fluorescence signals and dissociation curves are analyzed by ABI 7900 SDS 2.3 software.

      Cell death determination The M30-Apoptosense ELISA (Peviva, USA) is a one step in vitro
      immunoassay for the quantitative determination of the apoptosis-associated CK18Asp396 (M30)
      neo-epitope in plasma. The concentration of the antigen that quantifies ongoing apoptotic
      cell death, is expressed as U/L.

      The M30-Apoptosense ELISA is used in combination with M65 ELISA (Peviva, USA), for the
      quantitative determination of total soluble CK18 released from dead cells (necrotic and
      apoptotic). The concentration of the antigen is expressed as U/L.

      PIIINP and VEGF determination Plasma PIIINP levels will be determined using ELISA kit (USCN,
      China). PIIINP levels are expressed as ng/mL as a marker of tissue collagen deposition and
      fibrosis. Serum VEGF will be determined as a marker of neo-angiogenesis and tissue
      re-modeling using an ELISA kit (Bender MedSystems, Austria), data being expressed in ng/L.

      Statistic Data will be analyzed using one-way ANOVA, paired t-test, Student's t-test for
      unpaired data, linear regression (SPSS, STATSDIRECT) with statistical significance set at p
      <0.05 (2-tailed). All comparison regarding coffee exposure and abstinence will be carried
      within patients and not within groups, using, as said, a pair data approach.
    

Intervention name(s): Coffee; coffee abstinence
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): Administration of 4 cups of coffee per day for 1 month; total abstinence for both coffee and caffeine containing beverages
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: None (Open Label)
Primary purpose: Prevention