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Cyclosporine for Recurrent Hepatitis C after Liver Transplant

Permanent Link: http://ufdc.ufl.edu/UFE0024885/00001

Material Information

Title: Cyclosporine for Recurrent Hepatitis C after Liver Transplant A Randomized Pilot Study
Physical Description: 1 online resource (31 p.)
Language: english
Creator: Firpi-Morell, Roberto
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: cyclosporine, hepatitis, immunosuppression, liver
Clinical Investigation (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: CYCLOSPORINE FOR RECURRENT HEPATITIS C AFTER LIVER TRANSPLANT: A RANDOMIZED PILOT STUDY Cyclosporine is an immunosuppressant drug with antiviral activity in vitro against hepatitis C. We performed a pilot study to prospectively determine the safety, tolerability, and influence of cyclosporine vs. tacrolimus on viral response during therapy with Pegylated Interferon PEG (alfa)-2a and ribavirin in liver transplant recipients with recurrent hepatitis C infection. Thirty-eight patients were enrolled and randomized to continued tacrolimus or switched to cyclosporine at the University of Florida. Patients received PEG (alfa)-2a and ribavirin for 48-weeks if they have genotype 1, or 24 weeks if genotype 3. From the 38 patients, 20 patients received tacrolimus and 18 cyclosporine with a mean age of 53. Eighty-two percent were men, 84% Caucasian and 90% genotype 1. Sustained viral response for patients on cyclosporine was higher than in patients on tacrolimus receiving PEG/RBV therapy. This randomized-controlled pilot study is the first in vivo study evaluating cyclosporine vs. tacrolimus in liver transplant recipients undergoing antiviral therapy. Change from tacrolimus to cyclosporine led to a modest hepatitis C RNA drop and appeared to enhance the antiviral response of PEG (alfa)-2a and ribavirin, but this was not statistically significant. A larger randomized study will be necessary to determine if cyclosporine offers an advantage over tacrolimus in post transplant patients.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Roberto Firpi-Morell.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Asal, Nabih R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024885:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024885/00001

Material Information

Title: Cyclosporine for Recurrent Hepatitis C after Liver Transplant A Randomized Pilot Study
Physical Description: 1 online resource (31 p.)
Language: english
Creator: Firpi-Morell, Roberto
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: cyclosporine, hepatitis, immunosuppression, liver
Clinical Investigation (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: CYCLOSPORINE FOR RECURRENT HEPATITIS C AFTER LIVER TRANSPLANT: A RANDOMIZED PILOT STUDY Cyclosporine is an immunosuppressant drug with antiviral activity in vitro against hepatitis C. We performed a pilot study to prospectively determine the safety, tolerability, and influence of cyclosporine vs. tacrolimus on viral response during therapy with Pegylated Interferon PEG (alfa)-2a and ribavirin in liver transplant recipients with recurrent hepatitis C infection. Thirty-eight patients were enrolled and randomized to continued tacrolimus or switched to cyclosporine at the University of Florida. Patients received PEG (alfa)-2a and ribavirin for 48-weeks if they have genotype 1, or 24 weeks if genotype 3. From the 38 patients, 20 patients received tacrolimus and 18 cyclosporine with a mean age of 53. Eighty-two percent were men, 84% Caucasian and 90% genotype 1. Sustained viral response for patients on cyclosporine was higher than in patients on tacrolimus receiving PEG/RBV therapy. This randomized-controlled pilot study is the first in vivo study evaluating cyclosporine vs. tacrolimus in liver transplant recipients undergoing antiviral therapy. Change from tacrolimus to cyclosporine led to a modest hepatitis C RNA drop and appeared to enhance the antiviral response of PEG (alfa)-2a and ribavirin, but this was not statistically significant. A larger randomized study will be necessary to determine if cyclosporine offers an advantage over tacrolimus in post transplant patients.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Roberto Firpi-Morell.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Asal, Nabih R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024885:00001


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1 CYCLOSPORINE FOR RECURRENT HEPATITIS C AFTER LIVER TRANSPLANT: A RANDOMIZED PILOT STUDY By ROBERTO J. FIRPI MORELL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIR EMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Roberto J. Firpi Morell

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3 To my family

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4 TABLE OF CONTENTS P age LIST OF TABLES ................................ ................................ ................................ ........................... 5 LIST OF FIGURES ................................ ................................ ................................ ......................... 6 LIST OF ABBREVIATIONS ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ................................ ..... 8 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................... 9 2 MATERIALS AND METHODS ................................ ................................ ........................... 11 Study Design ................................ ................................ ................................ ........................... 11 Entry Criteria ................................ ................................ ................................ .......................... 11 Sample Size ................................ ................................ ................................ ............................ 12 Randomization ................................ ................................ ................................ ........................ 12 Immunosuppression ................................ ................................ ................................ ................ 13 Interferon B ased T herapy ................................ ................................ ................................ ....... 13 H epatitis C RNA Testing ................................ ................................ ................................ ........ 14 Histology ................................ ................................ ................................ ................................ 14 End Point ................................ ................................ ................................ ................................ 15 Statistical Analysis ................................ ................................ ................................ .................. 15 3 RESULTS ................................ ................................ ................................ ............................... 16 Characteristics of the Patients ................................ ................................ ................................ 16 Virologic Response (Serum HCV RNA) ................................ ................................ ................ 16 Serum Alanine Aminotransferase ................................ ................................ ........................... 17 Histological Analysis ................................ ................................ ................................ .............. 17 Adverse Event s ................................ ................................ ................................ ....................... 18 4 DISCUSSION ................................ ................................ ................................ ......................... 23 5 FUTURE WORK ................................ ................................ ................................ .................... 26 LIST OF REFERENCES ................................ ................................ ................................ ............... 27 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 30

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5 LIST OF TABLES Table page 3 1 Baseline characteristics of the pa tients ................................ ................................ ............. 21 3 2 Rates of adverse events, dose reduction, and discontinuation of treatment ....................... 22

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6 LIST OF FIGURES Figure page 3 1 Flo w diagram of study participants ................................ ................................ ................... 20 3 2 Viral response rates ................................ ................................ ................................ ........... 21

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7 LIST OF ABBREVIATIONS CsA Cyclosporine EVR Early v iral r esponse ETR End of treatment r esponse HCV Hepatitis C LT Liver t ransplant NR Non responder PEG Pegylated i nterferon RT PCR Reverse t ranscription p olymerase c hain r eaction SVR Sustained virological response TAC Tacrolimus

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8 Abstract of Th esis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CYCLOSPORINE FOR RECURRENT HEPATITIS C AFTER LIVER TRANSPLANT: A RANDOMIZED PILOT STUDY By R oberto J. Firpi Morell August 2009 Chair: Nabih R Asal Major: Medical Sciences Clinical and Translational Science Cyclosporine is an immunosuppressant drug with antiviral activity in vitro against hepatitis C. We performed a pilot study to prospectiv ely determine the safety, tolerability, and influence of c yclosporine vs. t acrolimus on viral response during therapy with Pegylated Interferon [ PEG (alfa) 2a ] and ribavirin in liver transplant recipients with recurrent hepatitis C infection. Thirty eight patients were enrolled and randomized to continued tacrolimus or switch ed to cyclosporine at the University of Florida Patients received PEG (alfa) 2a and ribavirin for 48 weeks if they have genotype 1, or 24 weeks if genotype 3. From the 38 patients, 20 patients received tacrolimus and 18 cyclosporine with a mean age of 53. Eighty two percent were men, 84% Caucasian and 90% genotype 1. Sustained viral response for patients on cyclosporine was higher than in patients on tacrolimus receiving PEG/RBV therapy This randomized controlled pilot study is the first in vivo study evaluating cyclosporine vs. tacrolimus in liver transplant recipients undergoing antiviral therapy. Change from tacrolimus to cyclosporine led to a modest hepatitis C RNA drop and appeare d to enhance the antiviral response of PEG (alfa) 2a and ribavirin but this was not statistically significant A larger randomized study will be necessary to determine if cyclosporine offers an advantage over tacrolimus in post transplant patients

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9 CHAP TER 1 INTRODUCTION A pproximately three percent is infected with Hepatitis C (HCV) and 3.2 million persons have chronic HCV infection in the United States (1) HCV infection may progress to end stage liver disease, the most common indication for liver transplantation (LT) in the United States. (2) Unfortunately, HCV recurrence is universal after LT and continues to be responsible for allograft failure in about ten percent of recipients by the fifth postoperative year (3) The increasing use of potent immunosuppressive agents and marginal (older) donors have been implicated in aggressive disease recurrence and graft loss. (4) Recurrence of HCV leads to a cumulative probability of clinical decompensation of 22% at one year, and 54% at three years after cirrhosis developed. (5) Unfortunately, due to very poor postoperative survival re transplantatio n is often not an option (6) In this study, we analyzed the impact of c yclosporine (CsA) immunosuppression on HC V replication and the response to interferon based therapy within our LT population. Cyclosporine and tacrolimus (TAC) are the two most frequently used immunosuppressive drugs in solid organ transplantation C sA has been shown to have antiviral activities (human immunodeficiency virus type I (7) herpes simplex virus (8) an d vaccinia virus (9) ) and recent studies have shown that CsA has a strong suppressive effect on HCV replication as well. (10) This CsA antiviral effect was found to be independent of its immunosuppressant function and was not noted with TAC. We have also demonstrated in a previous laboratory study that CsA suppresses HCV replication and viral nonstructural protein synthesis using a sub genomic HCV replicon system. (11) Nakagawa et al demonstrated in vitro that the HCV replication is mediated by blocking specific intracellular ligans of CsA known as cyclophilins. (12) Watashi and colleagues also demonstrated decrease of specific HCV proteins and HCV RNA in the replicon

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10 cell system. (10) Replication using the genotype 2a JFH1 strain was less sensitive to CsA showing the diverse effects of CsA on HCV. (13) This invitro data has led to clinical applications. CsA appears to have a beneficial impact on HCV antiviral therapy when combined with interferon. (14) Cyclosporine or TAC based immunosuppression does not appear to have a significant impact in the frequency or severity of recurrence HCV after LT. (15) However, long term studies in pati ents nave to antiviral therapy after LT have shown that patients on TAC have a higher biochemical recurrence (83%) compared to CsA (72%) in up to 37 months of follow up. (16) Histologic recurrence was also seen in 100 % of the patients on TAC vs. 87% of the patients on CsA. (16) Other groups have shown higher graft survival and lower incidence of fibrosing cholestatic hepatitis in LT recipients on CsA based immunosuppression. (17) However, several conflicting reports have also been published regarding the efficacy of CsA in the LT population; therefore, significant uncertainty regarding the potential role of CsA in the HCV transplant recip ient remains. (18 21) We hypothesize that CsA may help achieve a higher sustained viral response in patients recei ving antiviral therapy for recurrent HCV after LT The results of this pilot study would be used as hypothesis generating data and to power future studies to assess the influence of CsA vs. TAC on viral response in treatment of post transplant HCV.

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11 C HAPTE R 2 MATERIALS AND METHOD S Study Design This is a randomized, single center controlled study comparing two different immunosuppression regimens (CsA and TAC) in patients with recurrent HCV after LT undergoing antiviral therapy for HCV. Only adult HCV LT rec ipients who were nave or who have never received interferon based therapy following LT were enrolled in this study. The duration of the study was 72 weeks for genotype 1 (48 weeks of treatment and 24 weeks of follow up), and 48 weeks for genotype 3 (24 we eks of treatment and 24 weeks of follow up). The study was conducted at the University of Florida in Gainesville between July 2004 and April 2008. Patients attended clinic visits at the time of randomization (baseline) and at 12 weeks intervals for 72 week s. All other forms of liver disease (e.g., alcoholic cirrhosis, hepatitis B virus, autoimmune hepatitis, and genetic diseases) were excluded from our study population. All patients gave written informed consent before the initiation of the study. The study was approved by the Western Institutional Review Board at Olympia, WA. Entry Criteria At our center, patients undergo protocol liver biopsies to assess for recurrence of disease and staging after LT for HCV. All patients who were transplanted for HCV were considered eligible for screening using the following inclusion criteria: 1) males and females age 18 years or older able to provide a written informed consent 2) willing to practice acceptable birth control during the study period 3) elevated aminotran sferases (ALT), 4) detectable HCV RNA by polymerase chain reaction (PCR), 5) a liver biopsy after LT consistent with chronic HCV The biopsy must have Ishak Stage 2 to be considered for treatment Patients were excluded if they have previously received a ny interferon based therapy after LT, had decompensated cirrhosis, hemoglobin <12g/dl,

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12 WBC<3,500/mm 3 platelets < 75,000/mm 3 Human immunodeficiency virus infection, pregnancy, positive HbsAg, history of coronary artery disease, history of seizure disorder poorly controlled autoimmune conditions, thyroid dysfunction, diabetes mellitus, major psychosis, intolerance to previous interferon based therapy other than anemia or neutropenia, history of suicidal ideation or suicidal attempts, creatinine > 2.0 mg/dl severe non hepatic illnesses. Documentation of acceptable contraception in sexually active men and women of childbearing potential was required during treatment period and for 6 months after discontinuation of the drugs. Sample Size A power analysis was conducted for the primary hypothesis based on differences in SVR between CsA and tacrolimus in our preliminary data. Given a mean SVR difference of 20 30%, we will be able to detect mean differences with 110 patients per group; 10 additional subjects added to compensate for drop out at alpha=0. 05 (two tailed) at a power of .80. (SPSS version 1 6.0 for Windows, SPSS Inc., Chicago, IL). Overall, we estimate d that approximately 38 subjects randomized between the two arms would be a reasonable pilot study to he lp test the superiority of CsA in this population. Randomization Participants were assigned to interventions by using random allocation. Randomization was performed using computer generated random numbers. One hundred and fifty patients were assessed for eligibility (111 were excluded from the analysis mainly because they did not have an Ishak Stage 2) and 39 met entry criteria for enrollment in the study. Subjects with HCV recurrence (Ishak Stage 2) were enrolled and randomized to stay on TAC or to c hange to CsA for baseline immunosuppression with 1 month washout period before initiation of therapy with PEG 2a and ribavirin for 48 weeks for genotype 1, or 24 weeks for genotype 3. HCV RNA was assessed at entry, week 12, 24, 48, and six months post tre atment. Liver biopsies were

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13 performed at inclusion and at end of treatment. Proper allocation concealment was followed. The patients were not blinded to group assignment once they were randomized to one of the immunosuppressants. Immunosuppression Standar d immunosuppression at the time of transplantation consisted of TAC in combination with prednisone. Patients receiving TAC were treated with a dose of 0.08 0.12 mg/kg/day orally in two divided doses with target trough whole blood concentrations of 10 15 ng /ml for the first month post transplant followed by 5 10 ng/ml thereafter. Immunosuppression was typically tapered to monotherapy (TAC alone) within 4 6 months of transplantation. Prednisone was discontinued between month 4 and 6 of transplantation as per our transplant protocol for HCV patients. Patients randomized to CsA had TAC discontinued and were treated with CsA at a dose of 2.0 4.0 mg/kg/day orally in two divided doses with target trough whole blood concentrations of 150 200 ng/ml. There was no diff erence in the average steroid dose between the two groups. Interferon B ased T herapy Combination therapy with interferon and ribavirin was utilized in those patients with significant fibrosis (Ishak fibrosis stage > 2) on protocol or indication liver biops y. Therapy was initiated at a modified dose [PEG interferon alfa 2a 135 g and ribavirin 400 600 mg daily] for 2 weeks, and if tolerated, the dose was increased to full dose [PEG interferon alfa 2a 180 g weekly and ribavirin 800 1,200 mg daily]. Ribaviri n dosage was based on weight; patients < 75 kg received 800 mg and > 75 kg 1000 mg. Hemoglobin, white blood cell count, and platelet count were monitored weekly for the first four weeks and then monthly thereafter. Dose reduction was performed as follows: if PMN < 750 or platelets < 35,000/ L, the interferon was

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14 reduced one level; if PMN < 500 or platelets < 25,000, therapy was stopped; hemoglobin < 10 mg/dl, ribavirin was reduced to 600 mg per day; if hemoglobin < 8 mg/dl the ribavirin was discontinued. Er ythropoietin was used to prevent ribavirin discontinuation when possible. Neupogen was also allowed to treat neutropenia to avoid discontinuation. Therapy was discontinued in any patient who developed moderate to severe rejection, systemic bacterial infect ion, severe neuropsychiatric symptoms, or symptomatic anemia. The intended duration of treatment was 48 weeks for genotype 1 and 24 weeks for genotype 3. Hepatitis C RNA Testing Serum HCV RNA values were measured at entry into study, one month after switch to CsA (washout period), Day 0 of treatment, 12 weeks, 24 weeks, 48 weeks or end of treatment (ETR) and 6 months after completion of interferon based therapy to assess for the presence of a sustained vir a l response (SVR) in those patients with a negative HCV at ETR The HCV RNA titer was determined using a branched DNA signal amplification assay (Quantiplex HCV RNA; Chiron Corporation, Emeryville, CA). Serum samples that tested negative by the branched DNA assay were analyzed using RT PCR with a sensitivit y of about 10 200 copies (Qualitative Amplicore HCV Test; Roche, Mississauga, Ontario, Canada). Histology Protocol liver biopsies were performed in our institution at month 4, yearly or when clinically indicated after transplant. Recurrent HCV disease was scored for inflammation and fibrosis, using the modified Knodell scoring system of Ishak. (22) Patient s treated were those with Ishak score 2. Liver biopsies were repeated at the end of treatment for evaluation of histological changes after compl etion of treatment.

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15 End Point The primary end point was to perform a pilot study to determine the effect of CsA in viral clearance in the LT recipients with recurrence HCV during therapy with combination of interferon and ribavirin. Statistical A nalysis D ata are presented as median (range) for continuous variables and as frequencies for categorical variables. Comparisons between the two groups (SVR vs. NR) were performed with Pearson chi square tests or Fisher's exact test for categorical variables, and Ma nn Whitney U test for continuous variables. Statistical analysis was performed with statistical software (SPSS 16.0 for Windows 2007 ; SPSS Inc., Chicago, IL).

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16 CHAPTER 3 RESULTS Characteristics of the Patients The primary analysis was intention to treat and involved all patients who were randomly assigned to intervention. Of the 1,146 adult liver transplants performed at our institution from 1991 to 2006, 474 transplants were performed for the indication of HCV cirrhosis. Nineteen patients were randomize d to receive CsA and 20 patients were randomized to continue TAC as immunosuppressant regimen. All patients were nave to interferon based therapy after LT. F igure 3 1 illustrates the flow of participants through each stage of the study. (23) One patient randomized to receive CsA was not included in the analysis because he/she never received CsA or IFN RBV due to a pulmonary complication prior to dosing. The baseline characteristics were similar in both groups except for the mean baseline HCV RNA was higher in the CsA group (Table 3 1). Mean immunosuppression levels for TAC and CsA were maintained between 8 10 ng/ml for TAC and 100 150 ng/ml for CsA for the duration of the therapy. Virologic Response (Serum HCV RNA) Prior to initiation of PEG HCV RNA levels in the CsA group decreased by a mean of 0.39 2 million IU/ML (range 1.07 7.68)(p=0.5). Early vir a l response (EVR), defined as reduction of HCV RNA level by at least 2 log IU by week 12 was achieved by 11 (55%) of the patients in the TAC group vs. 13 (72%) in the CsA group (p=0.2). Serum HCV RNA became undetectable at 12 weeks in 10 (50%) and 9 (50%) of the TAC and CsA arm s respectively. Patients that di d not achieve the goal of at least 2 log drop in HCV RNA were considered non responders (NR) and treatment was discontinued. End of treatment response or undetectable virus at the end of therapy was achieved by 9 (45%) of the patients in the TAC group and 9 (50%) of the patients in the CsA

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17 group (p=0.7). The relapse rate was 22% for both groups TAC and CsA. In an intention to treat analysis of patients receiving at least one injection of IFN, 7 patients out of 20 (35%) in TAC vs. 7 out of 18 CsA patients (39%) (p=0.8) achieved a SVR (Figure 3 2) For the CsA group, those patients that achieved SVR had a significant lower baseline HCV RNA (p=0.02) and lower BMI (p=0.04) compared to non responders. In the subgroup of patients with genotype 1 (17 patients in each arm), EVR was achieved by 9 (53%) patients on TAC and by 12 (71%) in those on CsA ( p =0. 3 ). Seven (41%) in TAC and 8 (47%) in CsA achieved an ETR (p=0.4). Relapse rate was 14% (1 out of 7) and 25% (2 out of 8) in the TAC and CsA groups, respectively (p =0.6). There was not a statistically significant difference in SVR between the groups, 5 (31%) in TAC group vs. 6 (35%) in CsA group (p=0.5). Three patients were genotype 3 in the TAC arm (1 NR, 1 Relapse, and 1 SVR) and 1 patient in the CsA arm (1 SVR). Serum Alanine Aminotransferase Mean serum alanine aminotransferase concentrations (IU/mL) at ETR and end of follow up were 52 137 and 27 16 in the CsA arm vs. 52 30 and 53 41 in the TAC arm. There was no statistically significant difference betwe en the two groups. Interestingly, patients on the TAC group who achieved an SVR had a significant lower baseline ALT compared to the NR group (p=0.01). Histological Analysis Pretreatment and end of treatment liver biopsies were available in 33 (89%) of t he patients treated. Two biopsies were not available for 2 patients in each arm. The biopsies not available were the ETR biopsies from patients NR to treatment. The mean change in hepatitis activity index (HAI) demonstrated an improvement in both groups w ith a change of 0.88 units in the CsA group vs. 1.52 units in the TAC group (p=0.1). The mean change in fibrosis stage was +

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18 0.11 in CsA vs. 0.25 in the TAC group (p=0.09), mainly driven by those patients who had an SVR. Histologic improvement was a ssociated with SVR regardless of the treatment arm. Patient non responders to therapy had no change or increase in fibrosis score of 1 or 2 stages. Adverse Events Three patients had to discontinue therapy during the study in the CsA arm and 2 patients in t he TAC arm. Patients in the CsA arm discontinued therapy before the 48 weeks for the following reasons: one patient (eventual relapser) stopped at week 34 due to significant neutropenia, one patient (NR) stopped at week 36 due to anemia, and another patien t (NR) stopped at week 8 due to acute cellular rejection after IFN therapy was initiated. Patients in the TAC arm discontinued therapy before the 48 weeks for the following reasons: one patient stopped at week 38 due to high blood pressure and increase in liver function tests, the other patient stopped at week 37 due to severe fatigue and itching. Seven patients, 3 in TAC and 4 in CsA developed acute cellular rejection. From the patients that have acute cellular rejection on TAC, 2 achieved SVR and 1 was n on responder with negative virus at 12 weeks, but positive at 6 months. From the CsA group, 1 patient achieved SVR, 1 r elapse d 1 was a non responder with EVR, and one patient that achieved EVR died due to the development of chronic cellular rejection afte r IFN was initiated. Most of these episodes of acute cellular rejection were associated with lower than expected immunosuppressant levels in the presence of IFN therapy. Dose reduction of PEG IFN was required in 17 (94%) of the patients in the CsA arm and 12 (60%) in the TAC arm. This reduction was mainly secondary to neutropenia or thrombocytopenia (p=0.02). Dose reduction of RBV was necessary in 16 (89%) and 18 (90%) of the CsA and TAC group, respectively (p=1.0). Anemia was the most common reason for RBV dose reduction (Table 3 2). Fatigue, depression and headache were the most common adverse events seen in both groups.

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19 Growth factors were allowed in our study based on the type of population we were treating. Epoietin was used in 61% and 75% of the patie nts on CsA and TAC, respectively (p=0.4). Neupogen was also necessary in 50% and 35% of the patients on CsA and TAC, respectively (p=0.5).

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20 Figure 3 1. Flow d iagram of s tudy p articipant s. This figure shows the flow of study participants for each group (CsA vs TAC) in the study. TAC, tacrolimus; CsA, cyclosporine Assessed for eligibility, (n=150) Excluded, (n= 111) Not meeting inclusion criteria, (n=108) Refused to participate, (n=3) Other reasons, (n=0) Analyzed, (n=20) Excluded from analysis, (n=0) Lost to follow up, (n=0) Discontinue d intervention, (n=0) Allocated to intervention TAC, (n=20) Received allocated intervention, (n=20) Did not receive allocated intervention, (n= 0) Lost to follow up, (n=0) Discontinued intervention, (n= 1) (Chronic rejection) Allocated to intervention CsA, (n=19) Received allocated intervention, (n=18) Did not receive allocated intervention, (n= 1) (pulmonary complications prior to dosing) Analyzed, (n=18) Excluded from analysis, (n=1) Pa tient did not received intervention Allocation Anal ysis Follow Up Enrollment Randomized (n=39)

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21 Figure 3 2. Viral r esponse r ates. In an intention to treat analysis this figure shows the respo nse rate (EVR, ETR, SVR and Relapse) for the study group. TAC, tacrolimus; CsA, cyclosporine Table 3 1 Baseline c haracteristics of the p atients Characteristic Cyclosporine Tacrolimus Male sex, n (%) 13(72) 18(90) Mean age SD, yr 52.2 6.4 54.4 5.4 Race (C/AA/H), (%) (89/0/11) (80/10/10) Mean body mass index SD, kg/m 2 28.1 5.6 32 7.9 HCV G enotypes n (%) 1 17 (94.4%) 17 (85%) 3 1 (5.6%) 3 (15%) Baseline HCV RNA, IU/ ml 7.9 x 10 6 3.3 x 10 6 Alanine Aminotransferase SD, IU/ ml 107.1 70 .3 142.9 115.5 Hepatitis Activity Index SD 4.6 1.4 4.3 1.6 Fibrosis Score (Ishak) SD 2.8 1.0 2.2 0.6 n number; SD, s tandard d eviation ; yr, year; C, Caucasian; H, Hispanic; AA, African American; IU International Units 55% 72% 45% 50% 10% 11% 35% 39% 55% 72% 45% 50% 10% 11% 35% 39%

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22 Table 3 2. Rates of a dverse e vents, d ose reduction, and d iscontin uation of t reatment Variable Cyclosporine Tacrolimus P value Discontinuation 6% 0% 0.47 Rejection 22% 15% 0.7 Chronic cellular rejection 6 % 0% 0.47 Dose reduction 94% 95% 1.0 Interferon reduction 94% 60% .02 Ribavirin reduction 89% 90% 1.0 Neutropenia (<750 mm3) 39% 15% 0.1 Anemia 78% 90% 0.4 Thrombocytopenia 56% 55% 1.0 Adverse events Headache 33% 30% 1.0 Fatigue 44% 60% 0.5 Myalgia 11% 15% 1.0 Nausea 28% 29% 0.7 Fever 22% 25% 1.0 Insomnia 28% 50% 0.2 Irritability 11% 15% 1.0 Depression 39% 50% 0.5

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23 CHAPTER 4 DISCUSSION Hepatitis C after LT continues to be a challenge to the transplant community due to detrimental effects on bo th graft and patient survival. The factors that define disease progression are probably multifactorial, but donor age, steroids for early rejection, and HCV status before LT are among a few identified as important factors involved in this process. Neverthe less, t he impact of the primary immunosuppression regimen on disease and treatment outcomes is not well defined. A few in vitro studies have shown significant advantage of CsA over TAC in HCV anti viral activity in the replicon cell culture system. Nakagaw a et al treated HCV replicon cells with CsA showing suppression of viral replication, while Watashi and colleagues treated HCV replicon cells with CsA and showed decrease in both HCV proteins and HCV RNA levels. (10, 12, 24) In our lab, we confirmed previous studies showing a dose dependent anti viral activity against HCV, independent of classic IFN pathways. (11) In the clinical setting, we also found patients treated for HCV recurrence with IFN based therapy had a better chance of achieving a n SVR if they were on CsA for baseline immunosuppression, compared to TAC. (11) Anecdotal data from other HCV experienced centers have suggested similar findings. Thus we designed our trial as the first prospective randomized controlled study after LT to compare TAC vs. CsA in patients treated with PEG IFN and RBV. Th is first in vivo human study to examine the d ifference between CsA and TAC in patients treated with PEG IFN and RBV has two main findings. First, when patients were switched from a stable TAC dose to CsA, patients experienced a modest decrease in HCV RNA levels, suggesting some antiviral benefit to t his switch Secondly, the antiviral response to IFN RBV therapy appeared to be improved with the CsA based regimen, although this small pilot study was not powered to show significant SVR differences. We learned from this study that CsA may

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24 play a benefici al role as primary immunosuppression for patients transplanted for HCV infection and may offer an advantage over TAC in those patients undergoing IFN based therapy. Despite a recent study by Martin et al. showing no significant differences between TAC and CsA on histologic HCV recurrence after LT, this earlier study was not intended to show difference between the two, when patient s were on IFN based therapy. (25) Previous data f rom Japan showed that a combination of IFN and CsA may increase the activity of IFN against HCV (14) but no published data exists about the effect of CsA on HCV infected patients receiving treatment after LT. In our randomized study, patients on CsA receiving combination therapy with I FN viral response ; however no statistically significant differences were found between patients taking CsA vs. TAC Interferon use after LT for HCV recurrence has not consistently been associated with acute cellular rejection in most clinical studies. The rate of rejection varies substantially between studies, ranging from a 17 % 30% chance of acute cellular rejection with IFN therapy. (26 29) Rejection rate in our study was similar to previous reports in post transplant HCV recurrence. Interestingly 7 (18%) patients developed acute cellular rejection while on therapy. Three patients on TAC had reject ion, 2 of them achieved SVR despite the episode of acute cellular rejection and one was after an initial EVR. Of the 4 patients with acute cellular rejection in the CsA group, 1 patient achieved SVR, 2 patients had an EVR (complete EVR is now defined as HC V RNA negative) but were non responders at end of treatment, and 1 patient relapsed. Therefore, most of the rejection episodes occurred in patients while on treatment with PEG IFN and negative HCV RNA. One of the most intriguing finding s of this study is that most of the patients with acute cellular rejection achieved an EVR or SVR. Potentially as HCV is cleared from the liver, improved drug metabolism may le a d to lower CsA or TAC levels. Indeed, many

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25 of these rejections occurred in the setting of low lev els of immunosuppression, supporting this concept. In addition, HCV can lead to significant impairment of both the innate and adaptive immune response, leading to enhanced immune activity with viral clearance. (30) Given our experience in this trial, we now run TAC or CsA levels at higher doses in the setting of HCV anti viral therapy (TAC levels 7 10 ng/ml and CsA levels 150 200 ng/ml ). The small sample of patients is clearly the biggest limitation of this study. Our study did not have enough power to detect such difference giv en the limited sample size. Another limitation of the study is that we did not randomize CsA vs. TAC at time of transplant, but at the time of significant HCV recurrence (Stage 2) which may underestimate the impact of CsA. In our study, histological impr ovement was associated with an SVR regardless of the treatment arm. Therefore, antiviral therapy not only helps to achieve a sustained response but also to decrease inflammation and fibrosis in a population at high risk of rapid fibrosis progression. In a natural history study after LT, Clark et al found that IFN treatment reduces the risk of decompensation in patients who achieved an SVR. (5) Therefore, even if advanced disease is already present our results strengthen the argument for aggressive treatment of those with recurrent HCV af ter LT

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26 CHAPTER 5 FUTURE WORK Previous studies have show n that CsA anti HCV activity does not involve blockade of post translational proteins or IFN genes, and does not have a relationship with calcineurin/NFAT mediated pathway or immunosuppressive prope rties However, the anti HCV action is most probably via blockade of cellular CsA binding proteins, called cyclophilins. (12) These molecules may constitute novel targets for anti HCV therapeutics. Moreover, i n the pre LT popul ation the use of CsA is less attractive due to its immunosuppressive effects. The application of non immunosuppressive cyclophilin inhibitors with potent ability to suppress HCV, like NIM811 (Novartis Pharmaceuticals) or Debio 025 (DebioPharm), open the wa y for a novel approach to anti HCV treatment that could be complementary to IFN based treatment and also to future treatments such as protease and polymerase inhibitors. This first in vivo study does not suggest a potential advantage of CsA in HCV patient s undergoing antiviral therapy but definite conclusions based on this pilot study are not possible The results of this study can be used as hypothesis generating data and to power future l arger randomized controlled studies to further investigate these f indings.

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27 LIST OF REFERENCES 1. National Center for Disease Control and Prevention. Hepatitis C Fact Sheet. 2009 2. Belle SH, Beringer KC, Detre KM. Recent findings concerning liver transplantation in the United States.Los Angeles: UCLA Tissue Typing Laboratory, 1996. 15 29. 3. Gane EJ, Naoumov NV, Qian KP, Mondelli MU, Maertens G, Portmann BC, et al. A longitudinal analysis of hepatitis C virus replication following liver transplantation. Gastroenterology 1996 Jan;110(1):167 177. 4 Machicao VI, Krishna M, Bonatti H, Aqel BA, Nguyen JH, Weigand SD, et al. Hepatitis C recurrence is not associated with allograft steatosis within the first year after liver transplantation. Liver Transpl 2004 May;10(5):599 606. 5. Clark VC, Firpi RJ, So ldevila Pico C, Morelli G, Cabrera R, Levy C, et al. The Natural History of HCV related Cirrhosis After Liver Transplantation. 48 ed. 2008. S3. 6. Roayaie S, Schiano TD, Thung SN, Emre SH, Fishbein TM, Miller CM, et al. Results of retransplantation for re current hepatitis C. Hepatology 2003;38(6):1428 1436. 7. Wainberg MA, Dascal A, Blain N, Fitz Gibbon L, Boulerice F, Numazaki K, et al. The effect of cyclosporine A on infection of susceptible cells by human immunodeficiency virus type 1. Blood 1988 Dec;72 (6):1904 1910. 8. Vahlne A, Larsson PA, Horal P, Ahlmen J, Svennerholm B, Gronowitz JS, et al. Inhibition of herpes simplex virus production in vitro by cyclosporin A. Arch Virol 1992;122((1 2)):61 75. 9. Damaso CR, Keller SJ. Cyclosporin A inhibits vaccin ia virus replication in vitro. Arch Virol 1994;134((3 4)):303 319. 10. Watashi K, Hijikata M, Hosaka M, Yamaji M, Shimotohno K. Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes. Hepatology 2003 Nov;38(5):1282 1288. 1 1. Firpi RJ, Zhu H, Morelli G, Abdelmalek MF, Soldevila Pico C, Machicao VI, et al. Cyclosporine suppresses hepatitis C virus in vitro and increases the chance of a sustained virological response after liver transplantation. Liver Transpl 2006 Jan;12(1):51 57. 12. Nakagawa M, Sakamoto N, Tanabe Y, Koyama T, Itsui Y, Takeda Y, et al. Suppression of hepatitis C virus replication by cyclosporin a is mediated by blockade of cyclophilins. Gastroenterology 2005 Sep;129(3):1031 1041. 13. Ishii N, Watashi K, Hishik i T, Goto K, Inoue D, Hijikata M, et al. Diverse effects of cyclosporine on hepatitis C virus strain replication. J Virol 2006 May;80(9):4510 4520.

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28 14. Inoue K, Sekiyama K, Yamada M, Watanabe T, Yasuda H, Yoshiba M. Combined interferon alpha2b and cyclospo rin A in the treatment of chronic hepatitis C: controlled trial. J Gastroenterol 2003;38(6):567 572. 15. Hilgard P, Kahraman A, Lehmann N, Seltmann C, Beckebaum S, Ross RS, et al. Cyclosporine versus tacrolimus in patients with HCV infection after liver tr ansplantation: effects on virus replication and recurrent hepatitis. World J Gastroenterol 2006 Feb 7;12(5):697 702. 16. Villamil F, Levy G, Grazi GL, Mies S, Samuel D, Sanjuan F, et al. Long term outcomes in liver transplant patients with hepatic C infect ion receiving tacrolimus or cyclosporine. Transplant Proc 2006 Nov;38(9):2964 2967. 17. Rayhill SC, Barbeito R, Katz D, Voigt M, Labrecque D, Kirby P, et al. A cyclosporine based immunosuppressive regimen may be better than tacrolimus for long term liver a llograft survival in recipients transplanted for hepatitis C. Transplant Proc 2006 Dec;38(10):3625 3628. 18. Everson GT. Impact of immunosuppressive therapy on recurrence of hepatitis C. Liver Transpl 2002 Oct;8(10 Suppl 1):S19 S27. 19. Ghobrial RM, Colquh oun S, Rosen H, Hollis P, Ponthieux S, Pakrasi A, et al. Retransplantation for recurrent hepatitis C following tacrolimus or cyclosporine immunosuppression. Transplant Proc 1998 Jun;30(4):1470 1471. 20. Guitard J, Sandres Saune K, Kamar N, Ribes D, Faguer S, Esposito L, et al. Hepatitis C virus viral load after conversion from tacrolimus to cyclosporine in liver transplant patients: a pilot study. Transplant Proc 2007 Oct;39(8):2603 2605. 21. Teixeira R, Papatheodoridis GV, Burroughs AK. Management of recur rent hepatitis C after liver transplantation. J Viral Hepat 2001 May;8(3):159 168. 22. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1991 Oct;22(6):696 699. 23. Altma n DG, Schulz KF, Moher D, Egger M, Davidoff F, Elbourne D, et al. The revised CONSORT statement for reporting randomized trials: explanation and elaboration. Ann Intern Med 2001 Apr 17;134(8):663 694. 24. Nakagawa M, Sakamoto N, Enomoto N, Tanabe Y, Kanaza wa N, Koyama T, et al. Specific inhibition of hepatitis C virus replication by cyclosporin A. Biochem Biophys Res Commun 2004 Jan;313(1):42 47. 25. Martin P, Busuttil RW, Goldstein RM, Crippin JS, Klintmalm GB, Fitzsimmons WE, et al. Impact of tacrolimus v ersus cyclosporine in hepatitis C virus infected liver transplant recipients on recurrent hepatitis: A prospective, randomized trial. Liver Transpl 2004 Oct;10(10):1258 1262.

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29 26. Chalasani N, Manzarbeitia C, Ferenci P, Vogel W, Fontana RJ, Voigt M, et al. Peginterferon alfa 2a for hepatitis C after liver transplantation: two randomized, controlled trials. Hepatology 2005 Feb;41(2):289 298. 27. Eguchi S, Takatsuki M, Soyama A, Hidaka M, Tokai H, Hamasaki K, et al. Intentional conversion from tacrolimus to cy closporine for HCV positive patients on preemptive interferon therapy after living donor liver transplantation. Ann Transplant 2007 Oct;12(4):11 15. 28. Stanca CM, Fiel MI, Kontorinis N, Agarwal K, Emre S, Schiano TD. Chronic ductopenic rejection in patien ts with recurrent hepatitis C virus treated with pegylated interferon alfa 2a and ribavirin. Transplantation 2007 Jul 27;84(2):180 186. 29. Walter T, Dumortier J, Guillaud O, Hervieu V, Paliard P, Scoazec JY, et al. Rejection under alpha interferon therapy in liver transplant recipients. Am J Transplant 2007 Jan;7(1):177 184. 30. Rosen HR. Transplantation immunology: what the clinician needs to know for immunotherapy. Gastroenterology 2008 May;134(6):1789 1801.

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30 BIOGRAPHICAL SKETCH Dr. Roberto J. Firpi is an A ssistant P rofessor of M edicine in the University o f Florida Division o f Gastroenterology where he serves as the p rogram d irector of the Transplant Hepatology Fellowship Program for the Section of Hepatobiliary Diseases and Liver Transplantation. He received his medical degree from the University o f Puerto Rico School o f Medicine in 1994, completed a r esidency in i nternal m edicine at the University of Puerto Rico where he also served as Chief Resident, and obtained fellowship training in g astroentero logy and h epatology at the University of Florida under an NIH T32 t raining g rant In addition, he received his Master of Science in Medical Sciences with a concentration in clinical and translational research in 2009. Dr Firpi also completed additional t raining in h epatology and l iver t ransplantation at Cedars Sinai Medical Center in Los Angeles, CA where he received the AASLD is h epatology with an emphasis on the manage ment of liver transplantation, viral hepatitis and immunosuppression after liver transplantation. Dr. Firpi was appointed as Program Director of the Transplant Hepatology Fellowship Program and successfully completed an application for an ACGME approved t r ansplant h epatology f ellowship at the University of Florida, one of only 21 ACGME accredited programs in the U nited S tates Dr. Firpi has strong clinical and translational research interests, with more than five active clinical trials and multiple translat ional research initiatives underway. He has an extensive interest in chronic hepatitis C infection and liver transplantation and is currently applying for NIH funding through a career development award. In the short period of time as junior faculty at the University of Florida, Dr. Firpi has published multiple papers in the area of hepatitis C and liver transplant and has become a leader in the area. He serves as a reviewer for several journals including Liver Transplantation, American Journal of Gastroent erology American Journal of

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31 Transplantation, Journal of Hepatology and Transplantation He has presented his research annually at national meeting such as DDW, AASLD and the European International Meetings (EASL). He has also been a visiting professor a t national and international universities, including University of North Carolina, University of Puerto Rico, Oregon Health & Science University, St Luc Hospital in Montreal Canada, Toronto General Hospital, University of Alberta Hospital and others Dr F irpi has been the course director for the Annual Viral Hepatitis and Liver Disease Symposium for the State of Florida and currently is on the Advisory Board for the ALF and a member of the Membership Committee for the American Association for the Study of Liver Diseases. His accomplishments thus far are a reflection of not only mentoring and current research institutional environment, but also to his abilities to master the fundamentals of clinical investigation, grant writing and clinical study design. H e has mentored residents and fellows during his time at UF. He was awarded with the Exemplary Teacher Award of the Society of Teaching Scholars and also with the Faculty Award for e xcellence in teaching, role model and mentor by the C ollege of Medicine at the University of Florida.