Following liver transplantation (LT), graft reinfection by hepatitis C virus (HCV) is almost inevitable, but the clinical outcome of graft reinfection is variable. The impact of different immunosuppressive regimens on graft outcome following HCV reinfection remains poorly defined. Recent studies have observed that more recently transplanted HCV patients appear to be experiencing more aggressive graft reinfection with rapid progression to cirrhosis.1 In addition to advancing liver donor age,2 it has been suggested that changes in the use of immunosuppression may explain the inferior outcome for more recently transplanted patients.3 Previously, it was observed that a progressively lower dose of tacrolimus (TAC) is required post-LT to maintain a constant tacrolimus level in patients transplanted for HCV.4 However, to better understand the relationship of TAC blood levels to recurrent HCV infection, appropriate comparison groups should also be studied. Appropriate comparison groups would include patients receiving TAC who had undergone LT for a non-HCV, preferably nonrecurring, liver disease. In addition, the specificity of the observed relationship of HCV to TAC immunosuppression can be examined by examination of a cohort of HCV patients receiving alternative, for example cyclosporine A (CYA), immunosuppression.
In this study, we have examined TAC and CYA doses and blood levels in patients transplanted for HCV infection and in a comparison group transplanted for alcoholic liver disease (ALD). Thus, doses and levels were examined in 4 distinct patient groups from our center: HCV patients receiving TAC, HCV patients receiving CYA, ALD patients receiving TAC, and ALD patients receiving CYA.
All patients underwent LT at the Queen Elizabeth Hospital Liver Unit in Birmingham between 1990 and 2003. Consecutive patients transplanted for HCV and surviving at least 12 months after LT were identified from the Liver Unit database. For each patient, a comparison ALD patient was chosen from the database. The ALD patient undergoing transplantation following each HCV patient, also surviving at least 12 months and matched for age (within 5 years) and gender, was chosen for the comparison cohort. For some HCV patients, an appropriate comparison patient could not be identified.
The dose and blood levels of TAC and CYA in transplanted HCV patients (TAC, n = 44; CYA, n = 60) during the first 12 months after transplantation were analyzed. A comparison group of transplanted ALD patients (TAC, n = 44; CYA, n = 47) was also examined. All patients attended the Liver Unit outpatient clinic for follow-up. In general, following discharge from hospital after LT, patients were seen weekly for 1 month and then monthly for the reminder of the first post-LT year. At each attendance, routine blood tests included measurement of full blood count, serum electrolytes, and liver function tests. In addition, TAC and CYA trough levels were measured. Data were assessed at the following time-points: 2 weeks, months 1 to 6, and months 9 and12. For each patient at each time-point, the ratio of dose to blood level was also calculated.
In our center, standard immunosuppression (for first graft with no significant renal dysfunction) for most patients comprises corticosteroids, azathioprine, and a calcineurin inhibitor (CNI). The CNI dose is adjusted according to trough levels, and also considers a history of rejection, the presence of concurrent infection, renal dysfunction, or other signs of toxicity.
TAC dose at 0.1 mg/kg/day (0.05 mg/kg twice daily) is started initially by nasogastric tube, and then orally. In the stable patient, the TAC dose is adjusted to achieve 12-hour postdose levels of 10-15 ng/mL during the first month, 7-12 ng/mL during months 2 and 3, and 5-10 ng/mL after 3 months. During the first 3 months posttransplantation, the TAC level is maintained at the levels recommended above unless there is significant renal dysfunction, other toxicity requiring a dose reduction, or an episode of rejection necessitating a dose increase.
CYA is usually introduced at 4 mg/kg twice daily. For CYA, the target initial trough level during the first 3 months was 200-250 ng/mL, thereafter it was 150-200 ng/mL.
Azathioprine 1.5-2 mg/kg body weight is the standard second-line immunosuppressive agent and is commenced on the day of LT. Conversion to mycophenolate mofetil (starting dose of 0.5-1.0 g twice daily) is principally undertaken for patients who develop early significant renal function, and it is then used with low-dose TAC or CYA.
Where possible, drugs affecting cytochrome p450 (CYP3A4) were avoided.
During the years observed in this retrospective study, there was an evolution of Liver Unit protocol from the use of CYA (pre-1995) to the use of TAC (post-1995) as primary immunosuppression. Thus, the CYA- and TAC-treated patients did not contemporaneously undergo transplantation. Instead, the majority of TAC-treated patients underwent transplantation more recently than the comparison CYA cohort. However, as stated above, for each of the TAC and CYA cohorts, HCV and ALD patients underwent transplantation contemporaneously. For those few patients who changed from CYA to TAC, or vice versa, only those data collected after LT but before conversion were included in the analysis. In keeping with the date of LT, CYA patients were more likely to have received azathioprine, and TAC patients were more likely to have received mycophenolate mofetil as a second immunosuppressive agent. During the period of observation, the protocol for administration of corticosteroids was unchanged. That is, starting dose of prednisolone was 20 mg/day, weaned completely during the first 3 post-LT months.
Data were collected from the Liver Unit transplant database and from the hospital laboratory reporting system. Data were examined at the following specified time-points after LT: 2 weeks, months 1 to 6, and months 9 and 12. Of course, most patients did not attend clinic on those exact dates post-LT. To be eligible for inclusion in the analysis, data had to be available for a date within 10 days (either side) of the specified dates. When there were 2 or more attendances within 10 days of the specified date, the first available visit following the specified date was used.
To examine the possible effect of liver graft dysfunction on TAC and CYA doses and levels, additional analyses were made. Though crude, we defined graft dysfunction as a serum bilirubin >40 μmol/L after the second post-LT week (thus permitting time for bilirubin to settle after very early graft dysfunction and/or rejection).
We also reviewed the histology at 1 year after LT for HCV patients, and compared the pathology observed for TAC-treated versus CYA-treated HCV cohorts.
A total of 104 HCV patients were selected (TAC, n = 44; CYA, n = 60). The age- and gender-matched comparison group of ALD patients comprised TAC (n = 44) and CYA (n = 47) patients.
Table 1. Demographic Data of 104 HCV Post–Liver Transplantation Patients and 91 ALD Liver Transplantation Patients
Abbreviations: ALD, alcoholic liver disease; CYA, cyclosporin A; F, female; HCV, hepatitis C virus; M, male; TAC, tacrolimus.
Total number of patients
Age [years; median (range)]
Azathioprine treatment (number of patients)
Mycophenolate mofetil treatment (number of patients)
TAC Dose and Levels in HCV and ALD Patients
Despite a protocol that proscribed TAC levels greater than 10 ng/mL during the early post-LT months, the majority of TAC-treated patients had levels less than 10 during that period. During the 12-month period of observation, TAC levels were significantly higher for HCV than for ALD patients (P = 0.002) (Fig. 1A). For ALD patients, TAC dose and blood level remained fairly closely linked during the observation period. In contrast, for HCV patients it was observed that blood levels rose during the first months after LT, despite a significant reduction in TAC dose. The dose of TAC decreased over time for both HCV and ALD patients (P < 0.001), but the reduction was greater for HCV patients (P = 0.03) (Fig. 1B)
CYA Dose and Levels in HCV and ALD Patients
CYA dose decreased over time for both groups (P < 0.001) but a greater reduction was observed for the HCV group (P = 0.007) (Fig. 2B). For both HCV and ALD patients, CYA levels decreased over time (P < 0.001) but there was no significant difference between HCV and ALD patients (Fig. 2A). Thus, to maintain comparable blood levels, a greater reduction of dose was required for HCV than for ALD patients.
Ratio of Dose to Level for TAC and CYA in HCV and ALD Patients
We also calculated and compared the dose to level ratios at each time point for both TAC and CYA in both the HCV and ALD groups. This ratio provides an indication of the dose of drug required to achieve a given blood level. For TAC, the dose:level ratio is significantly greater for ALD than for HCV patients beyond 3 months after LT (Fig. 3). In other words, the dose of TAC required to achieve a given blood level of TAC diminishes during the 12-month period, but the diminution is greater for HCV than for ALD patients. For CYA, the dose:level ratio is also significantly lower for HCV than for ALD patients beyond 3 months after LT, but is statistically significant only between months 3 and 5. The ratio decreases during the year for HCV patients but there is no significant change for ALD patients.
We also assessed a possible impact of graft dysfunction on the TAC and CYA dose:level relationship. The above analyses were repeated for patients who had peak serum bilirubin greater than or less than 40 μmol/L beyond 2 weeks after LT. The observed relationships of CYA and TAC doses with levels in both HCV and ALD patients were not clearly different between the 2 groups (according to peak serum bilirubin).
Liver biopsy was performed at 1 year post-LT in 25 of 60 CYA-treated and 19 of 44 TAC-treated HCV patients. Fibrosis stage was reported according to the Ishak scoring system (0 to 6, 6 = cirrhosis).5 Fibrosis scores did not differ between the TAC- and CYA-treated HCV patients (Table 2).
Table 2. Fibrosis Staging of Patients at Year 1 for Tacrolimus- or Cyclosporin-Treated Hepatitis C Virus Patients
Fibrosis score (year 1 liver biopsy)
Tacrolimus (19 patients)
Cyclosporin (25 patients)
Published studies clearly show an impact of HCV infection on TAC and CYA levels in liver and kidney transplant patients.6–8 HCV infection appears to reduce the dose of TAC or CYA required to achieve a given blood level.
To examine the association of HCV with perturbation of TAC blood levels, we also examined a well-matched comparison group of patients who were transplanted for ALD. Though some patients resume alcohol intake after LT, few resume heavy alcohol intake during the first post-LT year, and the majority of ALD patients undergoing transplantation do not experience recurrent alcoholic liver damage. Thus, the impact of HCV recurrence and/or the associated liver inflammation on TAC metabolism could be studied. In addition, we compared both HCV and ALD TAC-treated patients with HCV and ALD CYA-treated patients.
The determination of TAC or CYA dose for a given patient is a complex process and is influenced by measurement of blood levels, by simultaneous assessment of liver and renal function, and by the use of concurrent second-line immunosuppressive drugs (principally azathioprine, mycophenolate mofetil, and corticosteroids). In general, higher doses of immunosuppression are used during the early post-LT weeks, and a gradual reduction is achieved during the first 12 months. This approach to immunosuppression is exemplified by examination of the whole-blood CYA levels measured for both HCV and ALD patients in this study. The median CYA levels for HCV and ALD patients were 220 and 230 ng/mL at 2 weeks post-LT and declined in a linear fashion for both groups during the following 12 months. For CYA-treated patients, the lower levels reflected a progressive reduction of CYA dose during the same period. For HCV patients, however, a significantly lower dose of CYA was required to achieve a given CYA blood level. Figure 3 demonstrates the difference in the CYA dose:level relationship for HCV versus ALD patients. For CYA-treated ALD patients, the ratio does not change significantly during 12 months. In contrast, for CYA-treated HCV patients, the ratio declines and is significantly different within 3 months of transplantation.
For TAC-treated HCV patients, a more disturbed relationship of dose to blood level is observed. However, the pattern observed for TAC-treated patients is similar to that observed for CYA-treated patients. The blood levels of TAC-treated ALD patients changed little during the 12 month period of observation. Surprisingly, the TAC levels rose for HCV patients, a change observed despite consistent and progressive reduction of the TAC dose. In other words, higher levels were observed despite lower doses. Again, the relationship of dose to blood levels is demonstrated in Fig. 3.
The dose:level ratio falls by greater than 50% for the HCV patients, but little change is seen for the comparison ALD group.
Hepatic injury/inflammation from recurrent HCV infection is associated with a strongly reduced requirement for TAC and the clearance of TAC decreases. As previously mentioned by Trotter et al.9 and van den Berg et al.,4 TAC, after absorption, is metabolized via hepatic cytochrome P4503A4 (CYP3A4) into different metabolites. TAC metabolites are subsequently excreted from the hepatocyte into the bile canaliculus by P-glycoproteins expressed on the hepatocyte membrane. Chronic HCV infection is associated with increased intrahepatic production of proinflammatory cytokines,10, 11 and it has been reported that CYP3A4 activity is down-regulated by these cytokines, such as interleukin 1, interleukin 6, and tumor necrosis factor α,12 and transforming growth factor β.13 Therefore, the higher level of tacrolimus in HCV patients may be due to impaired hepatic clearance of TAC, which may be either due to down-regulation of hepatic cytochrome CYP3A4 and/or impaired function of hepatic P-glycoprotein.
Our analysis of recurrent HCV infection on TAC and CYA dose and levels was not designed to assess the relative effects of those drugs on HCV-related liver damage. Nevertheless, it may be noteworthy that biopsy fibrosis scores assessed at 1 year post-LT did not apparently differ between CYA- and TAC-treated HCV patients.
The effect of HCV on TAC and CYA metabolism has potentially important clinical consequences. For LT recipients, recurrence of HCV infection, and/or the associated hepatitis, demand a regular adjustment of TAC dose to prevent inappropriately excessive immunosuppression and its consequences. In retrospect, our reaction to blood TAC levels for the HCV patients appears inadequate and delayed. Despite progressive reduction of TAC doses for the HCV patients, we observe a rise in blood levels, which return to below baseline levels at 12 months post-LT. Unintended potential consequences of these higher levels would include exacerbation of the HCV infection by augmented immunosuppression and undesirable toxic (mainly nephrotoxicity) and metabolic effects. Metabolic effects of TAC (and to a lesser extent, CYA) include insulin resistance and diabetes. The importance of both HCV per se and TAC (in comparison with CYA) for development of insulin resistance and new onset diabetes has been convincingly demonstrated in a number of studies and analyses.14–16 For instance, in a controlled study of CYA versus TAC for primary immunosuppression in LT patients, a significantly greater number of TAC patients developed diabetes.17 In a follow-up study of HCV patients from the same study, histological recurrence was worse in the TAC- versus the CYA-treated patients.18 It is well recognized that insulin resistance is an important determinant of histological progression in HCV infection. Thus, as far as possible, high levels of CNIs (particularly TAC) should be avoided for HCV patients. Vicious cycles could be established. HCV recurrence could lead to high TAC levels, high TAC levels could promote HCV replication and contribute to insulin resistance and diabetes, and histological progression could be accelerated.
In addition to the potential for high drug levels due to HCV infection, clinically relevant changes in blood levels may be observed when HCV is eradicated by antiviral therapy. Blood TAC (and CYA) levels for a given dose have been seen to fall following HCV suppression, and graft rejection can supervene as a direct consequence.
All of these observations suggest that cytochrome p450 3A4 may be inhibited in the HCV-infected liver. Our study and other published studies cannot, however, confidently distinguish the direct effects of HCV infection per se from the effects of the inflammatory response. HCV reinfection of the graft occurs immediately after LT. It has been shown that blood HCV titer returns to pre-LT levels within days of transplantation.19 Biochemical evidence of graft hepatitis is typically delayed until at least 1 month post-LT. Our study has made few observations in the early post-LT period. The complexity and many determinants of graft dysfunction in the early post-LT period would make it difficult to interpret the impact of early events on the dose:level relationship. We have attempted to explore the possibility that the decline in the dose:level ratio is a simple consequence of liver dysfunction secondary to HCV infection. We found no association of severity of graft dysfunction (as reflected by the serum bilirubin) with the dose:level relationship.
In vitro studies have shown that proinflammatory mediators such as interleukins and tumor necrosis factor can down-regulate cytochrome p450 function. Thus, it seems likely that the hepatitis associated with HCV infection causes cytochrome inhibition with an effect on TAC and CYA metabolism. The effect is reversed when the HCV-associated inflammatory response is eliminated by antiviral therapy.
In summary, our study supports and extends the observations of others in a large suitably controlled population of LT patients. These observations demand careful and frequent scrutiny of CNIs during the development of graft hepatitis and during antiviral treatment of HCV.