IMPROVEMENT OF CARDIOVASCULAR RISK FACTORS AND COSMETIC SIDE EFFECTS IN KIDNEY TRANSPLANT RECIPIENTS AFTER CONVERSION TO TACROLIMUS

Aims: Tacrolimus and Cyclosporine A (CyA) are cornerstones in immunosuppressive therapy. Cyclosporine side eff ects include hypertension and hypercholesterolemia both of which may increase the risk of cardiovascular mortality, gingival hyperplasia and hirsutism are known to reduce quality of life. The aim of this prospective study was to evaluate changes in cardiovascular risk profi le and cosmetic side eff ects after conversion from CyA to tacrolimus. Methods: 25 stable kidney transplant recipients (9 male, 16 female) were converted from a CyA to a tacrolimus – based regimen. Mean age was 45.7 ± 13.5 years. Time to switch following transplantation was 4.7±1.7 years. Reasons for conversion were multiple: arterial hypertension (9), hypertrichosis (3), gingival hyperplasia (3), hyperlipidemia (14). Results: 19/25 patients completed the one year study period. One patient died, two returned to hemodialysis, two were switched back to CyA and one patient was lost to follow-up. There were statistically signifi cant changes (p = < 0.05) in systolic and diastolic pressure and antihypertensive medication could be reduced in 13 patients. The dose of lipid-lowering agents could be reduced in the majority of the recipients and a complete withdrawal was achieved in 7 patients. Hypertrichosis and gingival hyperplasia resolved in all patients. Further, there was a signifi cant improvement (p = <0.05) in urea and serum creatinine levels. Adverse events were consistent with the established safety profi le for tacrolimus. Conclusions: Conversion to a tacrolimus-based regimen led to an improvement in the cardiovascular risk profi le. Further, cosmetic side eff ects which may lead to non-compliance, resolved after the switch.


INTRODUCTION
Cyclosporine and tacrolimus are calcineurin inhibitors, which are fundamental for currently used immunosuppressive protocol after transplantation.Cyclosporine and tacrolimus are eff ective in reducing the incidence of acute rejection.Both drugs have a similar safety profi le but they diff er with regards to the mechanism of action, safety profi le, toxicities and side eff ects 1,2 .
Well-known side eff ects of CyA therapy are nephrotoxicity, arterial hypertension, hyperlipidemia, gingival hyperplasia and hypertrichosis 3 .Hyperlipidemic and hypertensive eff ects of CyA increase the risk of posttransplant cardiovascular disease and as important nontraditional risk factors contribute to the high cardiovascular morbidity and mortality in renal transplant patients 4 .Hyperlipidemia and hypertension are also risk factors for chronic transplant nephropathy and reduce long term graft function and survival 5,6 .Gingival hyperplasia and hypertrichosis are known to reduce quality of life and may lead to non-compliance 7 .
The above mentioned negative side eff ects associated with CyA can be substantially reduced by switching to tacrolimus based therapy.Results from previous studies suggest that tacrolimus is associated with less hypertension and hyperlipidemia 8,9 .Following conversion to tacrolimus also excellent results have been described in the resolution of gingival hyperplasia and hypertrichosis 10 .
The aim of this 12 months, open, prospective, single arm, one-centre study was to evaluate the effi cacy and safety of the conversion from a CyA-to a tacrolimus-based immunosuppressive regimen in a group of stable kidney transplant patients suff ering from CyA related side eff ects.

PATIENTS AND METHODS
This was a prospective, nonrandomized, single -arm, one -center study which was conducted according to ICH/GCP guidelines in the University Hospital Olomouc.The trial was approved by the Local Ethics Committee and all patients gave written and dated informed consent before inclusion in the study.The endpoints of this study were to evaluate the impact of tacrolimus on cardiovascular risk factors, graft function and cosmetic side eff ects Subjects who met all of the following inclusion criteria were eligible for this study: Male or female patients, 18 years or older, patients with hyperlipidemia defi ned as fasting total cholesterol concentration in serum above 6.5 mmol/l or triglycerides serum concentration above 2.5 mmol/l, patients with arterial hypertension, systolic blood pressure ≥ 140 mm Hg or diastolic blood pressure ≥ 90 mm Hg despite treatment with at least one antihypertensive drug, gingival hyperplasia, hypertrichosis severe enough to require treatment, primary kidney transplantation or re-transplantation from a deceased donor and female patients of child bearing age who agreed to maintain eff ective birth control practice during the study.
Exclusion criteria included: Female patients who were pregnant or breast feeding, patients < 18 years of age, allergy or intolerance to HCO-60 or structurally related compounds, macrolide antibiotics or tacrolimus, patients required ongoing systemic immunosuppressive therapy other than for kidney transplantation, patients with present malignant disease, patients with signifi cant, uncontrolled concomitant infections and/or severe diarrhea, vomiting or active peptic ulcer, and patients participating in another clinical trial.Improvement of cardiovascular risk factors and cosmetic side eff ects in kidney transplant recipients after conversion to tacrolimus Before conversion the patients were treated with the well -established triple-drug maintenance immunosuppressive regimen consisting of CyA (Sandimmun Neoral) combined with mycophenolate mofetil (CellCept) and corticosteroids (Prednison).The doses of Sandimmun Neoral in these long-term transplant patients were adjusted to maintain target whole blood trough levels between 100-200 ng/ml.Cyclosporine levels were measured using FPIA (Fluorescence polarisation immunoassay), Abbott diagnostics.Doses of CellCept and Prednison were 20 mg//kg/day and 5 mg/day respectively.
Mean time to switch following transplantation was 4.7±1.7 years.Tacrolimus (Prograf) was started within 24 hours after patients received last dose of CyA.Initial daily dose of Prograf after switching was 0.1 mg/kg/day, divided into two doses.Target tacrolimus blood trough levels were 5-15 ng/ml at months 1 and 4-10 ng/ml by month 6.Tacrolimus levels were measured using MEIA (Microparticle enzyme immunoassay), Abbott diagnostics.No changes were made in concomitant immunosuppressive therapy after switch to tacrolimus.
Clinical status (physical examination, body weight, blood pressure, cosmetic side eff ects) and laboratory parameters (serum creatinine, plasma urea, aspartate aminotransferase-AST, alanine aminotrasferase-ALT, serum bilirubin, glycaemia, triglycerides, total cholesterol, high density lipoprotein cholesterol-HDL cholesterol, low density lipoprotein cholesterol-LDL cholesterol) were monitored weekly for the fi rst month after conversion and monthly thereafter.Blood pressure was measured in a sitting position after at least 5 minutes rest by certificated automated equipment.Blood samples for laboratory assessments were taken after overnight fasting and were measured at a certifi ed routine clinical chemistry laboratory on the Hitachi 917 analyzer (Roche).
All statistical analyses were performed by the linear regression analysis using data software system ADSTAT version 2.0 and QC EXPERT version 2.7.
Nineteen patients completed the one year study period.One patient died from acute myocardial infarction.Two patients had a substantially elevated serum creatinine levels at study entry and they had to go back to dialysis due to graft failure.Two patients were switched back to cyclosporine: One patient under tacrolimus treatment had diarrhoea and the second one decided to quit the medication as mild alopecia occurred.One patient was lost to follow-up.The available data of all 25 patients are summarized in Table 1 and Table 2.
Mean systolic blood pressure was reduced from 142.0 ± 21.4 mm Hg at baseline to 129.2 ± 13.9 mm Hg at week 52 (Fig. 1; regression curve y = 138-0.719x).Mean diastolic blood pressure was reduced from 84.0 ± 9.0 mm Hg at baseline to 81.2 ± 7.7 mm Hg at the end of one year observation (Fig. 2; regression curve y = 82.53-0.2x).Observed changes in systolic and diastolic blood pressure were statistically signifi cant (p<0.05) and antihypertensive medication could be reduced in 13 patients.
Mean serum total cholesterol level at baseline was 5.61 ± 1.19 and after conversion at week 52 was 4.68 ± 0.97 mmol/l (Fig. 5; regression curve y = 5.285-0.022x).Mean serum triglycerides levels before conversion was 2.27 ± 1.39 mmol/l and after switch to tacrolimus at week 52 was 2.13 ± 0.97 (Fig. 6; regression curve y = 2.000-0.005x).Mean LDL cholesterol level at baseline was 2.94 ± 0.96 mmol/l and at week 52 after switch 2.49 ± 0.68 mmol/l (Fig. 7; regression curve y =2.94-0.02x).Mean HDL cholesterol concentration was at the start of the study 1.39 ± 0.47 mmol/l and at the end of study 1.40 ± 0.52 mmol/l (Fig. 8; regression curve y = 1.397-0.012x).With the exception of HDL there was a trend towards lower values for these parameters however the diff erences did not reach statistical signifi cance.It is noteworthy that the dose of lipid-lowering agents could be reduced in the majority of the recipients and a complete withdrawal was achieved in 7 patients (50 % of hyperlipidemic patients).
Hypertrichosis and gingival hyperplasia resolved in all patients.No new onset or worsening of diabetes mellitus was observed after conversion.There were no signifi cant changes in fasting blood glucose concentrations during the study (5.56 ± 1.11 vs. 5.69 ± 1.00 mmol/l).No changes in liver function parameters were observed.Adverse events were consistent with the established safety profi le for tacrolimus.

DISCUSSION
Cyclosporine together with tacrolimus represent a group of calcineurin inhibitors which have the ability to inhibit calcineurin phosphatase, the essential enzyme in the process of the gene transcription for interleukin-2 (IL-2) and other proinfl ammatory cytokines.The inhibition of calcineurin phosphatase results mainly in suppression of T cell activation but it also suppresses molecules involved in cell-mediated immunity.The consequence of   this is interruption in the downstream sequence of events leading to allograft rejection.Despide this fact, there are many diff erences in action, safety profi le, toxicities and side eff ect between these two drugs 2,3,11 .Tacrolimus, in comparison with CyA signifi cantly reduces the incidence of acute rejection and improves survival of grafts as well as patients after kidney trans-plantation 12 .Long term monitoring of the result of the tacrolimus and CyA therapy has confi rmed, that tacrolimus-based immunosuppressive therapy signifi cantly reduces the risk of graft failure.According to the results of an American multicenter, comparative, randomized study the fi ve-year graft survival rate in the tacrolimus arm was 63.8 %, whereas in the CyA arm only 53.8 %.The diff er-Improvement of cardiovascular risk factors and cosmetic side eff ects in kidney transplant recipients after conversion to tacrolimus ence in the patients' survival rate in both groups, however, did not reach statistical signifi cance 13 .
Development of molecular-biological technique helps clarify the cause of the better immunosuppressive eff ectiveness of tacrolimus in comparison with CyA.A most important fi nding of immunological research and related to this, is that tacrolimus, unlike CyA, lowers mRNA expression for IL-l0, increases apoptosis of T lymphocytes activated by anti-gene, decreases production of antibody against HLA as well as HLA anti genes and signifi cantly decreases proliferation of vas intima 1,14,15 .
From the long-term point of view, particularly significant is the fact, that tacrolimus, unlike CyA, has no eff ects on levels of transforming β factor (TGF β), which has an important fi broproductive potential and is considered the key factor in the development of chronic transplant nephropathy and cosmetic side eff ects 16,17 .
Nonimmunological factors that are very important for graft and patient survival are hyperlipidemia and arterial hypertension.Hypertension after calcineurin inhibitors is caused by renal vasoconstriction and sodium retention.Long term CyA treatment in renal transplant patient resulted in higher renal vascular resistance and greater need for antihypertensive therapy compared with tacrolimus 18 .Hypertension and hyperlipidemia are common complications after renal transplantation and both are not only important determinants of graft function and graft survival, but also very important cardiovascular risk factors in the transplanted population.In general patients after kidney transplantation are at high risk of developing cardiovascular disease.Patients treated with CyA tend to have higher blood pressure, higher total cholesterol and LDL cholesterol levels and cosmetic adverse events -gingival overgrowth and hypertrichosis 12 .Tacrolimus is less nephrotoxic than CyA and has a more favourable cardiovascular risk profi le due to better graft function and better lipid and blood pressure profi le compared to CyA 4 .However, the incidence of glucose intolerance and postransplant diabetes mellitus is higher in tacrolimus treated patients 19 .
The results of our one-year study demonstrated, that following conversion from a CyA to a tacrolimus based immunosuppression protocol systolic and diastolic blood pressure were statistically signifi cant decreased and antihypertensive medication could be reduced in 13 patients.
The dose of lipid lowering agents could be reduced in the majority of the recipients and complete withdrawal was achieved in 7 patients (50 % of hyperlipidemic patients).Furthermore graft function improved, with a statistically signifi cant decrease in mean serum urea concentration and mean serum creatinine concentration.Hypertrichosis and gingival hyperplasia resolved in all patients.
In some aspects similar observations concerning the cardiovascular risk factors after renal transplantation and side eff ects of cyclosporine have been published in several other studies.In the European Tacrolimus vs. Cyclosporine Microemulsion Renal Transplant Study Group the authors found signifi cant lower cholesterol levels and mean arterial blood pressure in tacrolimus treated patients.The summary measure of serum triglycerides was not diff erent between treatment groups 20 .The better cardiovascular risk profi le of tacrolimus based immunosuppression was confi rmed in a 2 years follow-up of this study.Graft loss, patient death and biopsy-proven acute rejection occurred signifi cantly more frequently in patients treated with cyclosporin than in the tacrolimus arm.Graft function at 2 years post-transplantation was signifi cantly better and cholesterol and triglycerides concentrations were lower in the tacrolimus arm compared to the cyclosporine arm 21 .In the European Switch to Tacrolimus Study 296 adult kidney transplant patients with cyclosporine related side eff ects were converted to tacrolimus.Tacrolimus was well tolerated and the study demonstrated a substantial reduction in cardiovascular risk factors especially hyperlipidemia and arterial hypertension following the switch to tacrolimus-based therapy 22 .
In a 6 months randomised controlled conversion study of 124 renal transplant recipients published by M. Artz et al a signifi cant decrease in blood pressure, LDL cholesterol, serum apolipoprotein B and serum triglycerides was observed after conversion.Furthermore renal function improved 23 .A follow-up of 2 years in this study demon- strated that conversion from cyclosporine to tacrolimus was benefi cial to renal function, cardiovascular risk profi le and side eff ects.The Framingham risk score decreased from 5.7 ± 4.3 to 4.8 ± 5.3 (ref. 24).A signifi cant reduction in systolic blood pressure, serum total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides after conversion to tacrolimus were seen in a six months study published by Marcén 25 .No changes in diastolic blood pressure were observed in this study.In contrast to this, in an open-label single-arm prospective study in 22 adult renal recipients converted from cyclosporine to tacrolimus, signifi cant improvement in fi brinogen, total cholesterol and LDL cholesterol levels was observed but there were no signifi cant diff erences in HDL cholesterol, homocystein, C-reactive protein, haemoglobin A1c levels, serum creatinine, mean blood pressure and mean number of antihypertensive medication before and after conversion 26 .
Finally, in a one-year observation study of kidney allograft recipients converted from cyclosporine microemulsion to tacrolimus no changes in mean systolic and diastolic blood pressure were observed but signifi cant changes in creatinine levels, total cholesterol levels and LDL cholesterol levels were described 27 .
Well-known side eff ects of CyA treatment are hypertrichosis and gingival hypertrophy 10 .These complications may cause signifi cant morbidity and have psychological impact in transplant patients.Gingival hyperplasia may be associated with gum bleeding, pain and tooth loss.Pathogenesis of CyA induced gingival overgrowth is multifactorial.The dosage of CyA, duration of therapy as well as plasmatic and salivary concentrations of CyA play a central role.In addition, many other factors such as standards of oral hygiene, age, genetic predisposition, gingival infl ammation, some growth factors, especially TGF β and synergic or protective eff ects of other prescribed drugs contribute to the development of gingival hyperplasia 7,28 .
Gingival hyperplasia and hypertrichosis usually regressed completely within several months after switch from CyA to tacrolimus in the majority of patients effected.Residual gingival hyperplasia may persist in some individuals but is generally asymptomatic and requires no further specifi c dental treatment 7 .
In our study gingival hyperplasia and hypertrichosis signifi cantly or completely resolved in all patients converted to tacrolimus.The resolution of hypertrichosis and gingival hypertrophy improved body image, patient satisfaction and may lead not only to better quality of life, but also to better therapy compliance 29,30 .

CONCLUSION
The results of our study demonstrated, that conversion from CyA to tacrolimus in stable chronic transplant recipients was associated with signifi cant decrease of systolic and diastolic blood pressure (p<0.05).With respect to graft function urea and creatinine serum levels signifi cantly decreased (p<0.05).Conversion from cyclosporine to tacrolimus further allowed reduction of lipid-lowering agents in the majority of the recipients and a complete withdrawal was achieved in 7 patients (50 % of hyperlipidemic patients).These fi ndings confi rmed better cardiovascular risk profi le of tacrolimus based immunosuppression.Cosmetic side eff ects resolved after the switch, which may lead to better compliance and better quality of life.

Table l .
Evolution of blood pressure and urea and creatinine serum concentration.

Table 2 .
Evolution of parameters of lipid metabolism.