Mycophenolic acid trough levels after kidney transplantation in a cyclosporine-free protocol.

Twenty-seven stable kidney transplant recipients treated with cyclosporine and prednisone were converted to mycophenolate mofetil (MMF) and prednisone 1 year after transplantation. After conversion the patients were treated with a standard daily dose of 1 g MMF b.i.d. and 10 mg prednisone for 4 months. Thereafter, two MMF dose reductions were performed with a 4-month interval. Mycophenolic acid (MPA) trough levels were measured at regular intervals. A relation was found between MPA trough levels and MMF dose. The median MPA trough level for patients treated with 1 g MMF b.i.d. was 4.3 microg/ml (0.95-15.5) and 3.0 microg/ml (0.73-7.8) for patients treated with 750 mg b.i.d. (P = 0.0002). The MPA trough levels further decreased from 3.0 to 2.3 microg/ml (0.6-6.63) in patients treated with 500 mg MMF b.i.d. (P = 0.01). Dose reduction of MMF from 1 g to 750 mg b.i.d. could be performed without acute rejections. A further dose reduction to 500 mg b.i.d. elicited 3 rejections. Patients experiencing an acute rejection had a median MPA trough level of 2.3 microg/ml (1.26-3.38) compared to 3.8 microg/ml (1.48-6.52) in patients without an acute rejection (P = 0.25). We conclude that there is a significant relation between MPA trough levels and MMF dose. MPA trough levels were not predictive of rejection in the present study.

TNF-alpha: mRNA, plasma protein levels and soluble receptors in patients on chronic hemodialysis, on CAPD and with end-stage renal failure.

BACKGROUND: Patients on hemodialysis suffer from an impaired immunity against infectious agents, hyporesponsiveness to vaccination and are prone to develop malignancies. This clinical state of immunoincompetence may be due to a disbalance in their defense mechanisms in which TNF-alpha and its soluble receptors 1 and 2 play a central role. PATIENTS AND METHODS: We measured, with double-sandwich ELISA, the levels of TNF-alpha and the soluble TNF-receptors in peripheral blood of patients on chronic intermittent hemodialysis (CIHD), on peritoneal dialysis (CAPD) and pre-dialysis end-stage renal failure (ESRF). Using reverse transcriptase polymerase chain reaction (RT-PCR) analysis, we quantified the amount of TNF-alpha mRNA in peripheral blood mononuclear cells (PBMC) obtained from these patient groups. RESULTS: In none of the patient groups, elevated levels of TNF-alpha were detected with ELISA, while high levels of soluble TNF receptors were present in ESRF, CAPD and CIHD patients. This may be the result of an activated TNF-alpha system or due to their impaired renal clearance. TNF-alpha mRNA level was elevated in CIHD patients compared to ESRF and CAPD patients or healthy controls. CONCLUSION: This suggests that only during chronic HD is the TNF-alpha system activated. High levels of sTNFR, found in ESRF or CAPD patients do not reflect activation of TNF-alpha system, but are the result of impaired renal clearance of the receptors. Indeed, we found a strong linear correlation between the levels of sTNF receptors and renal function. Nevertheless, these high levels of sTNF receptors are biological active, as they were able to bind active TNF-alpha up to 75% (range 46 - 83%) and thus inhibit the bioactivity and bioavailability of produced TNF-alpha. This may play a role in the immunoincompetence of these patients.

Effect of cyclosporine on mycophenolic acid trough levels in kidney transplant recipients.

BACKGROUND: Triple drug treatment consisting of mycophenolate mofetil (MMF), in a standard dose of 2 g daily, combined with cyclosporine (CsA) and prednisone, has become the standard immunosuppressive regimen after kidney transplantation in many centers. The need for therapeutic drug monitoring of mycophenolic acid (MPA) has not yet been established. Several drug interactions with MMF are known. We investigated the influence of CsA withdrawal on MPA trough levels in renal transplant patients. METHODS: Fifty-two patients were treated with 1 g of MMF twice daily, and prednisone and CsA targeted between 125 and 175 ng/ml for 6 months after transplantation. At 6 months after transplantation, 19 patients were randomized for continuation of triple therapy (group A), 19 patients discontinued CsA (group B), and 14 patients discontinued prednisone (group C). We compared 12-hr fasted MPA trough levels at 6 and 9 months after transplantation within and between these groups. RESULTS: MPA trough levels during treatment with CsA, MMF, and prednisone were significantly lower than those during treatment with MMF and prednisone only (group B); median levels were 1.87 mg/L (range: 0.56-5.27) vs. 3.16 mg/L (range: 0.32-7.78), respectively (P=0.002). MPA trough levels in groups A and C did not change between 6 and 9 months after transplantation; group A median levels were 1.87 (range: 0.31-4.32) vs. 1.53 mg/L (range: 0.36-3.70), and group C median levels were 1.62 (range: 0.69-10.34) vs. 1.79 mg/L (range: 0.54-6.00), respectively. At 9 months after transplantation, patients in whom CsA was discontinued had higher MPA trough levels as compared with patients who continued the use of triple therapy (P=0.001) or patients in whom steroids were withdrawn (P=0.014). CONCLUSION: A significant increase of MPA trough levels was found after discontinuation of CsA (6 months after transplantation), resulting in almost a doubling of MPA trough levels at 9 months after transplantation. This resulted in increased MPA levels in patients without CsA as compared to MPA levels in patients continuing triple therapy or discontinuing prednisone.

Effect of mycophenolate mofetil on erythropoiesis in stable renal transplant patients is correlated with mycophenolic acid trough levels.

BACKGROUND: Both mycophenolate mofetil (MMF) and azathioprine (AZA) are immunosuppressive drugs that inhibit purine synthesis. In theory, MMF selectively inhibits lymphocyte proliferation, while AZA has well-known effects on red blood cells and thrombocytes as well. In renal transplant recipients we replaced CsA therapy by MMF in an attempt to reduce the immunosuppressive load 1 year after kidney transplantation. During this study we observed the effect of MMF on haematological parameters such as haemoglobin (Hb), leukocytes, and thrombocytes. METHODS: One year after kidney transplantation 26 stable patients were converted from cyclosporin A (CsA) to MMF (2 g/day). Thereafter, these patients were tapered twice in their MMF dose from 2 g to 1.5 g (4 months after conversion) and from 1.5 to 1 g (8 months after conversion) per day. The Hb levels, leukocyte and thrombocyte counts, and mycophenolic acid (MPA) trough levels were routinely measured. RESULTS: After conversion from CsA to MMF not only creatinine levels and the number of leukocytes, but also the haemoglobin (Hb) level significantly decreased in 21/26 patients (P=0.0004). In eight patients the Hb level dropped more than 1 mmol/l (=1.61 g/dl). Only in two of eight patients was an explanation for blood loss found. The effect on Hb level did not ameliorate after the first MMF dose reduction to 1.5 g/day. After tapering the MMF dose to 1 g/day, the Hb approached the pre-conversion level. Not only the MMF dose but also the mycophenolic acid (MPA) trough level correlated with the Hb level. CONCLUSIONS: After conversion from CsA to MMF 1 year after kidney transplantation, a decrease in Hb level and leukocyte count was observed. The MPA trough level correlated also with the Hb level. The effect on the Hb level was reversible after dose reduction. This finding suggests that MMF exerts a negative effect on erythropoietic cells.

The TNF-alpha system in heart failure and after heart transplantation: plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity.

BACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To assess the activity of the TNF-alpha system in patients with heart failure and after heart transplantation. METHODS: We measured TNF-alpha mRNA expression of peripheral blood mononuclear cells, plasma levels of TNF-alpha and sTNF reverse transcriptase receptors, using polymerase chain reaction and ELISA and performed a TNF-alpha binding capacity analysis, quantitating the buffer capacity of patients' plasma. RESULTS: In 11 patients with heart failure and in 15 cardiac allograft recipients, the TNF-alpha mRNA expression was comparable to controls. This level of mRNA was not accompanied by detectable TNF-alpha plasma levels. Significantly higher sTNF receptors levels were found in patients: ( P <0.001; ANOVA). The TNF-alpha binding capacity of patients' plasma was significantly increased, which led to decreased TNF-alpha recovery ( P<0.05). Both sTNF receptors showed a linear correlation with serum creatinine (sTNF-RI: r=0.92; sTNF-R2: r=0.82, P<0.001). CONCLUSIONS: The TNF-alpha mRNA expression and plasma levels show that the 'peripheral' TNF-alpha system is not activated. The high sTNF-receptors levels and their elevated TNF-alpha binding capacity, resulting in decreased TNF-alpha bioavailability, may contribute to an immunosuppressed state in these patients.

Mycophenolic acid plasma concentrations in kidney allograft recipients with or without cyclosporin: a cross-sectional study.

BACKGROUND: Combining cyclosporin (CsA) and prednisone with mycophenolate mofetil (MMF) results in a significant reduction in the rate of biopsy-proven acute rejection after kidney transplantation. This is achieved with a standard daily MMF dosage of 2 or 3 g. Whether monitoring of the pharmacologically active metabolite mycophenolic acid (MPA) will lead to improved safety and efficacy is unclear. METHODS: We monitored MPA trough levels in 18 kidney transplant recipients treated with CsA, prednisone, and MMF (63 samples) and in 11 patients (31 samples) treated with prednisone and MMF only, in a cross-sectional study. All patients were at least 3 months after transplantation with stable graft function. All patients were treated with 2 g MMF for at least 3 months and 10 mg prednisone. RESULTS: The MPA trough levels in the CsA-treated patients were significantly lower (P<0.0001; Mann-Whitney) than those in patients on MMF and prednisone only (mean MPA levels 1.98+/-0.12 vs 4.38+/-0.40 mg/l respectively). CONCLUSIONS: Although all patients were treated with an identical MMF dose, a significant difference was found in the MPA trough levels between CsA- vs non-CsA-treated patients. This suggests that CsA influences the MPA trough level. The level at which CsA affects the MPA trough levels is unclear.

The TNF-alpha system after successful living-related kidney transplantation.

The TNF-alpha system is thought to play a central role in the reduced immunity of haemodialysis patients. The imbalance between the high levels of soluble TNF receptors R1 and R2 and the low levels of immunoactive TNF-alpha results in an increased TNF-alpha buffering capacity leading to reduced immune responses. Apart from impaired renal clearance of the receptors, inefficient TNF-alpha production as a result of the uraemia may also contribute to the imbalance between this cytokine and its receptors. In patients receiving a living-related kidney transplant, renal function is nearly normalized in a very short period. This restoration of renal function may result in a state of better immunocompetence, either as a result of improved clearance of the receptors or as a result of reversal of the uraemic state. To differentiate between these two possibilities, we measured TNF-alpha protein, mRNA and the soluble TNF receptors R1 and R2 before and after successful renal transplantation. TNF-alpha mRNA was not affected by transplantation, indicating constant TNF-alpha production. The imbalance in the TNF-alpha system was markedly improved after transplantation, although normal values of the soluble receptors were not reached. One year after transplantation in stable kidney transplant recipients there was still an imbalance in the TNF-alpha system caused by persistently elevated levels of the soluble TNF-receptors. These results suggest that even after successful kidney transplantation the TNF-alpha system remains activated. However, despite immunosuppressive therapy, recipients of a living-related kidney do have a better balanced TNF-alpha system compared to haemodialysis patients.

Impact of continuous hemofiltration on cytokines and cytokine inhibitors in oliguric patients suffering from systemic inflammatory response syndrome.

The impact of continuous hemofiltration (CHF) using a polyacrylonitrile membrane on the kinetics of tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), and their inhibitors (soluble TNF receptors [sTNFrI, sTNFrII], interleukin-1 receptor antagonist [IL-1Ra]) was assessed in nine oliguric patients suffering from systemic inflammatory response syndrome. Blood and plasma flow (Qb, Qp), sieving coefficient (SC), plasma and ultrafiltrate clearances (Kp, Kuf), and plasma extraction rates (ERp) were calculated at different time points using standard formulas. No significant improvement of hemodynamics or gas exchange was noted following HF but a significant increase in serum bicarbonate occurred after 24 h (P < 0.05). TNF alpha was detected in plasma from all patients (153 +/- 2.3 pg/mL [mean +/- SEM]). None of the patients had detectable IL-1 beta levels. High levels of the TNF receptors (sTNFrI 20.338 +/- 2.431 pg/mL; sTNFrII 17.839 +/- 2.630 pg/mL) and IL-1Ra (19.775 +/- 3.943 pg/mL) were found in all patients. Upon initiation of hemofiltration (HF), the mean individual sTNFrI/TNF alpha ratio amounted to 269 +/- 84.6 and the sTNFrII/TNF alpha ratio to 249 +/- 91.8. Mean ultrafiltrate volume (Vuf) was 11.8 +/- 0.4 L/day. Appreciable sieving of IL-1Ra (SC 0.45 +/- 0.10), but not of the other cytokines, was noted (SC TNF alpha, sTNFrI, sTNFrII < 0.09). Despite minimal Kuf of TNF alpha, sTNFrI, and STNFrII (Kuf < 0.8 mL/min), appreciable Kp was noted, suggesting that membrane adsorption occurs (Kp approximately 8 mL/min). There was a nonsignificant increase of the ratios between both TNF receptors and TNF alpha across the filter (sTNFrI/TNF alpha ratio [pre] 231 +/- 37.9 versus [post] 312 +/- 75.3); sTNFrII/TNF alpha ratio [pre] 211 +/- 42.1 versus [post] 291 +/- 79.3). Appreciable Kp of IL-1Ra was noted (Kp 17.3 +/- 1.61 mL/min), which was only in part due to Kuf (4.0 +/- 0.86 mL/min). There was a significant decrease of IL-1Ra levels across the membrane, both overall ([pre] 20.223 +/- 2.282 versus [post] 16.637 +/- 2.039 pg/mL; P < 0.01) and at different time points (P < 0.01). Only for IL-1Ra was significant extraction from plasma noted (ERp 26 +/- 6.0%). Plasma levels of TNF alpha, sTNFrI, sTNFrII, and IL-1Ra were not altered by 24 h of CHF. In conclusion, both cytokines and cytokine inhibitors can be removed from the circulation, either by convective transport or by membrane adsorption. Using low-volume HF (Vuf approximately 12 L/day), no impact on cytokine plasma levels nor the patients hemodynamics or gas exchange was noted. The appreciable SC of IL-1Ra (0.45), however, suggests that HF with high(er) UF volumes (> 50 L/day) may be able to achieve reductions in plasma levels of some peptide (anti)mediators. However, whether this aspecific elimination of both mediators and antimediators may alter the clinical course in critically ill patients remains to be investigated.

A randomized trial comparing safety and efficacy of OKT3 and a monoclonal anti-interleukin-2 receptor antibody (BT563) in the prevention of acute rejection after heart transplantation.

In a prospective randomized trial, BT563, a murine IgG, anti-interleukin-2 receptor antibody, was compared with OKT3 for use as an early rejection prophylaxis after heart transplantation. Patients received either BT563 (n=31) or OKT3 (n=29) during the first 7 days after transplantation; cyclosporine was started on day 3. Median follow-up was 34 months. A cytokine release syndrome occurred in the majority of patients of the OKT3-treated group but in none of the BT563 recipients. The mean duration of electrical stimulation of the heart in the BT563 group was longer than in the OKT3 group (5.1 vs. 2.1 days). In both groups, one patient required insertion of a permanent pacemaker. Freedom from acute rejection at 3 months was not significantly different between the two groups (BT563: 5/29, 17%; OKT3: 6/29, 21%). In the BT563 group, however, rejection tended to occur earlier after transplantation. There was no difference in the overall incidence of rejection. The incidence of infectious complications was evenly distributed in both groups. Malignancies occurred in two patients, both in the OKT3 group. In conclusion, the use of this anti-interleukin-2 receptor monoclonal antibody in heart transplant recipients is safe and devoid of the side effects that accompany the use of OKT3. OKT3 and BT563 result in a similar freedom from rejection at 3 and 12 months after heart transplantation.

Pharmacodynamics of prophylactic antirejection therapy with an anti interleukin-2 receptor monoclonal antibody (BT563) after heart and kidney transplantation.

A mouse monoclonal antibody (BT563) directed to the alpha-chain of the IL-2 receptor was administrated immediately after transplantation in a dose of 10 mg/day prophylactically to 30 heart transplant recipients (HTx) and 40 renal transplant recipients (RTx) to induce immunosuppression. Plasma levels increased to a plateau level of 5300 ng/ml in HTx and 5900 ng/ml in RTx. BT563 plasma disappearance curves gives a mean T1/2 of respectively 39 h (range 14-112 h) and 42 h (range 8-122 h) for HTx and RTx respectively. The CD25 marker (IL-2R) on the peripheral blood lymphocytes disappeared within hours after the first gift and returned to normal within 0-20 days after the last gift. In HTx more often CD25+ cells were found in the presence of BT563 and more rejections occurred shortly after discontinuation of BT563 compared to the RTx group. Rejectors and non-rejectors within the HTX group did not differ with respect to the period of depletion of CD25 positive cells in the peripheral blood. In 56% of the patients a substantial IgM antibody response was detected. This response was similar for HTx and RTx and not related to rejection. The frequency of IgG responses was low in both HTx (13%) and RTx (21%) patients and the IgG response was not related with graft rejection or with antirejection treatment. Peripheral monitoring showed that mAb plasma levels, antimurine antibody responses and number of CD25 positive cells were not related with the clinical results. The mAb BT563 proved to be safe with respect to the generation of antimurine antibodies and, when given in combination with CsA, is a therapy with a potential for high efficacy.

A double-blind, placebo-controlled study of monoclonal anti-interleukin-2 receptor antibody (BT563) administration to prevent acute rejection after kidney transplantation.

In a double-blind, randomized, placebo-controlled trial, BT563, a murine IgG1 anti-IL-2R antibody, was given as a rejection prophylaxis after kidney transplantation. Drug-related side effects were not observed. During the 10-day course of BT563, no rejections (0/27) were found, whereas a rejection episode occurred in 7 patients (7/29) (P = 0.01) during placebo treatment. Within the first 4 postoperative weeks, freedom from rejection in the BT563 group and in the placebo group was 96% vs. 76% (P = 0.05). Due to rejection in the placebo group, 2 grafts were lost. At 3 months, an overall rejection incidence in the BT563 and placebo group was found of 3/27 (11%) vs. 8/29 (28%) patients (P = 0.18). Infectious complications were distributed equally between the 2 groups. CMV disease, found in 3 placebo-treated patients, occurred after rejection treatment (2/3). Within the BT563 group, 1 patient lost his graft due to renal artery thrombosis, 2 grafts were lost as a result of technical failure, and 2 patients had a squamous cell carcinoma that could be treated curatively. We conclude that the use of the anti-IL-2R mAb BT563 effectively prevents rejection after kidney transplantation without increasing infectious complications.

Intragraft monitoring of rejection after prophylactic treatment with monoclonal anti-interleukin-2 receptor antibody (BT563) in heart transplant recipients.

BACKGROUND: Anti-interleukin-2 receptor monoclonal antibodies have been used successfully in the prevention of rejection in cardiac allografts in several animal models. METHODS: In an open randomized study murine monoclonal CD3 antibody and BT563, a murine anti-interleukin-2 receptor monoclonal antibody, were given as rejection prophylaxis during the first week after heart transplantation. Cyclosporine therapy was initiated at the third postoperative day. RESULTS: In half the BT563-treated patients an early rejection was histologically shown at week 1, whereas heart transplant recipients treated with murine monoclonal CD3 antibody had a rejection incidence at week 1 of only 9%. During BT563 treatment CD25-positive cells (i.e., cells bearing the interleukin-2 receptor) were not detectable in peripheral blood. However, immunohistologic studies of endomyocardial biopsy specimens taken 1 week after transplantation showed the presence of CD25-positive cells within these specimens in 8 of 10 (80%) of patients with rejection. In patients without rejection CD25-positive cells were present in the biopsy specimens of only two of nine patients (22%). Reverse-transcriptase polymerase chain reaction studies on biopsy material showed the presence of messenger RNA for the interleukin-2 receptor in all and for interleukin-2 in three of five (60%) of biopsy specimens of rejecting grafts. CONCLUSIONS: Although CD25-positive cells were not detectable in peripheral blood during BT563 treatment, these cells were at the same time found to be present within 80% of the endomyocardial biopsy specimens from the rejecting grafts. By initiating cyclosporine treatment at day 0, the synergistic effect of combining cyclosporine and anti-interleukin-2 receptor monoclonal antibodies may result in a lower rejection incidence.

In vitro and in vivo effects of BT 563, an anti-interleukin-2 receptor monoclonal antibody.

BT 563, a murine anti-IL-2R MoAb, was found to be more potent than anti-Tac in inhibiting proliferation in the mixed lymphocyte reaction. Results obtained with 33B3.1 in these experiments were similar to those with BT 563. The anti-IL-2R MoAb 2A3 was shown to be a suitable agent for monitoring the effect of BT 563 on peripheral blood. IL-2R-positive cells were not detected in peripheral blood samples from 1 h after the first dose until 8 days after the last dose. Plasma trough levels were measured in patients receiving 5 or 10 mg daily. The administration of BT 563 to allograft recipients did not lead to clinically significant side effects.

Single-shot, high-dose rabbit ATG for rejection prophylaxis after kidney transplantation.

We studied the effects of a single intravenous injection of rabbit ATG (RIVM, Bilthoven, The Netherlands) in a dose of 8 mg/kg body weight administered 6 h after kidney transplantation on graft survival, rejection incidence, T-cell subsets, and cost-effectiveness. A total of 58 (37 male/21 female) consecutive renal allograft recipients were entered in this trial. Treatment results were compared with 56 patients treated with intravenous cyclosporin (CyA). In all patients concomitant medication consisted of steroids and azathioprine, followed by oral CyA. Following rabbit ATG, T cells (WT31) quickly disappeared from the peripheral blood and a return to greater than 100/mm3 was observed at a median of 7 (range 3-21) days. Graft survival was the same in both groups, as was the incidence of primary nonfunction. The rate of acute rejection was significantly lower in the rabbit ATG-treated patients (12% vs 50%). We conclude that a single shot of rabbit ATG is an attractive, easy, and cost-effective induction scheme with a low incidence of delayed graft function and acute rejection episodes. A relatively high incidence of vascular thrombosis of the graft, however, warrants further study before this treatment regimen can be generally applied.