CsA Levels Research Articles

CLINICAL BENEFIT OF NEORAL DOSE MONITORING WITH CYCLOSPORINE 2-HOUR PEAK LEVELS COMPARED TO TROUGH LEVELS IN STABLE LIVER TRANSPLANT PATIENTS

42 We have previously reported that cyclosporine (CsA) 2-hour peak levels(C2) correlate better with the area-under-the-curve than trough levels (C0) in heart Tx pts receiving Neoral. Moreover, Neoral dose adjustment according to a target CsA C2 blood level of 300-600 ng/ml(EMIT) resulted in 30% dose reduction and initial lower serum creatinine(Scr), without increasing the risk of acute rejection. It has also been reported that C2 levels correlate well with acute rejection after liver Tx. Purpose: to determine the clinical benefit of Neoral dose monitoring with conventional CsA C0 levels compared to 2 different ranges of C2 levels in stable liver Tx pts.Methods: clinical benefit was defined as the absence of acute rejection and no change in Scr at a mean follow-up period of 7 months. We enrolled 35 stable pts one year or more after Tx. There was no difference in primary liver disease, or any of the following baseline values: liver function tests, Scr, Neoral dose and number of pts on Neoral monotherapy. Pts were randomized into 3 groups (Gr) for Neoral dose monitoring: Gr 1(C0 target range 100-200 ng/ml): 7F/4M, 59±13 yr., 39±15 months post-Tx; Gr. 2 ("high" C2, target range 700-1000 ng/ml): 5F/8M, 58±11 yr., 35±17 months post-Tx, and Gr. 3 ("low" C2, target range 300-600 ng/ml): 3F/8M, 56±9 yr., 37±19 months post-Tx. Neoral dose was increased or decreased by 0.5-1 mg/kg/day if measured levels were outside of the target range. A liver biopsy was done if liver function tests increased >50% from baseline.Results: one pt in Gr. 1 and one pt in Gr. 3 presented with a reversible acute rejection episode within 1 month after enrollment; the CsA level of each of these had been below target. Neoral dose (Mg/kg/day), Scr levels, and mean percentage change are shown below.Table Conclusion: Neoral dose monitoring according to a CsA C2 range of 300-600 ng/ml resulted in a lower dose of Neoral and greater clinical benefit (stable Scr without a higher incidence of acute rejection) compared to conventional C0 levels or a higher C2 range in stable liver Tx pts.This research was funded by Novartis Pharma Canada inc.

CYCLOSPORINE LEVEL CHANGES IN PEDIATRIC RENAL TRANSPLANT (TX) PATIENTS DURING GROWTH HORMONE THERAPY.

57 Pediatric renal Tx patients with persistent growth failure may be treated with Growth hormone (GH). However, GH therapy has been associated with increases in serum creatinine levels and possible rejection. Recent studies in humans have revealed an increase in cytochrome P450 activity (CYP3A4 hepatic enzyme) with GH treatment. However, there have been no studies to examine the effect of GH treatment on Cyclosporine (CSA) metabolism. Therefore, 15 patients (12 boys and 3 girls) with 11 cadaveric and 4 living-related well-functioning grafts (ages from 5-14 yrs), who received GH therapy at least 1 year after renal Tx, were studied retrospectively. Measurements of whole blood 12 hour trough CSA levels by the TDx monoclonal assay were examined at the time of GH therapy initiation and at 3, 6, 9, and 12 months pre and post-GH therapy. The initial average CSA dose was 6.1±3.1 mg/kg/day; the average prednisone dose was 0.12±0.04 mg/kg/day. Other immunosuppressive therapy included lmuran (Azathioprine) at 1.2±0.2 mg/kg/day or Cellcept (Mycophenolate mofetil) at 35.3±12.1 mg/kg/day. GH therapy was discontinued in one patient after 4 months of treatment because of creatinine elevation and acute rejection, confirmed by renal biopsy. In another patient, GH treatment was discontinued after 11 months for acute rejection, also confirmed by renal biopsy. Of the remaining 13 patients, 9 patients completed 12 months of GH treatment, 3 patients completed 9 months and 1 patient completed 6 months of treatment. CSA dose adjustment to maintain appropriate immunosuppression was done when indicated. A significant decrease in CSA level (p< 0.05) was noticed at 3 and 12 months post-GH treatment compared with baseline, in the absence of a significant change in the weight-adjusted CSA dose in mg/kg/day. Paired Two-tailed Student t test was used (Table).Using ANOVA (single factor), CSA levels at 3, 6, 9 and 12 months during GH therapy were significantly lower than baseline, 3, 6, 9 and 12 months prior to GH therapy (p=0.0145), at a time when no difference in CSA dose was detected. These results suggest that GH treatment decreases CSA levels. Increasing CSA dosage may be needed during GH treatment to prevent acute or subclinical rejection.

DECREASED EXPRESSION OF TRANSFORMING GROWTH FACTOR-BETA (TGF-β) IN BIOPSIES WITH CHRONIC ALLOGRAFT NEPHROPATHY AFTER ONE YEAR OF 50% CYCLOSPORINE LEVEL REDUCTION AND MYCOPHENOLATE ADDITION

228 TGF-β is a growth-regulating protein that has been shown to enhance collagen production in vivo and in vitro. Overproduction of TGF-β can lead to renal damage by increasing fibrosis and may be important in the pathogenesis of chronic allograft nephropathy. High cyclosporine (CSA) levels might cause or worsen chronic renal injury by increasing TGF-β and collagen synthesis. Therefore, the current study was designed to determine the TGF-β1 isoform expression in human renal biopsy tissue by immunohistochemical staining (IHCS) in 5 patients with biopsy proven chronic allograft nephropathy and no evidence of acute rejection before and after a 50% reduction in CSA levels and addition of 1 g bid of mycophenolate mofetil(MMF). Interval between biopsies was 13.7 ± 2 months. Mean CSA levels were reduced from 200 to 100 ng/ml (12 hour trough TDX assay). TGF-β1 IHCS staining score (0-4) and positive staining area (+%) were significantly decreased in 4 of 5 patients with reduction in CSA levels. TGF-β1 predominately localized in the proximal tubular region, and was minimally evident in control biopsy specimens (IHCS 0.1-0.2).TableThese data suggest that reduction in CSA and addition of MMF may diminish production of TGF-β and result in decreased collagen synthesis and stabilization or improvement in fibrosis in kidney tissue of patients with chronic allograft nephropathy.

Total Plasma Homocysteine Concentrations are Strongly Correlated with Whole Blood Cyclosporine Levels and Angiographic Coronary Artery Disease in Heart Transplant Patients.

98 Introduction: Circulating total homocysteine (Hcy) levels are generally elevated in cardiac transplant patients. The etiology of elevated Hcy levels and its impact on transplant coronary artery disease (TxCAD) is unknown. Purpose: To identify clinical, biochemical and genetic factors that may influence hyperhomocysteinemia and to determine the association between Hcy and TxCAD in a cohort of cardiac transplant patients. Methods: 72 patients who underwent cardiac transplantation between 1984-1995 (mean 3.98 +/- 0.37 yr.) had the following parameters assessed; Hcy levels, age, gender, time after transplant, serum folate and B12 levels, total protein, urate, creatinine, albumin, and trough whole blood cyclosporine (CsA) levels. The presence or absence of angiographic TxCAD was assessed in the patients who were transplanted prior to 1995 (n=48). Results: All transplant patients had Hcy levels above our normal(>15uM). The mean Hcy level in the transplant group (25.4 +/- 0.8 uM) was significantly greater than in controls (8.3 +/- 2.1 uM, n=14, p<0.001), or in pt attending a high risk thrombosis clinic (16.8 +/- 1.3 uM, n = 94, p < 0.05). Serum creatinine was elevated (144 +/- 6 uM) and folate decreased (6.75+/- 0.22 nM) in the transplant patients. Multiple linear regression showed that creatinine (p=0.021) and through CsA levels (191 +/- 19 ug/L, p=0.015) were independent positive predictors of Hcy, while serum folate (p=0.018) and time since transplant (p=0.049) were significant negative predictors. Hcy was not associated with the prevalent methylenetetrahydrofolate reductase (MTHFR) polymorphism, A677V, even when interaction effects with serum folate were included in the regression model. The mean Hcy levels were higher in patients with angiographic TxCAD (25.96, n=25) than in those patients with normal coronary angiograms (21.89, n=23; p<0.05). Conclusions: Hyperhomocysteinemia is prevalent in cardiac transplant patients. Renal dysfunction and CsA levels have adverse effects on Hcy whereas high serum folate levels appear to be protective. Hcy levels were associated with an increase in angiographic TxCAD. Because folate levels were relatively low, supplementation with high dose folate may reduce the impact of Hcy on transplant coronary artery disease in these patients.

Coadministration of neoral and the novel rapamycin analog, sdzrad,to. lung allograft recipients:potentiation of immunosuppressive efficacy and reduction of toxicity by staggered vs. simultaneous treatment

518 Neoral and RAD are substrates for the same metabolic pathways and have complementary mechanisms for inhibition of lymphocyte proliferation. Therefore, we investigated treatment regimens designed to minimize pharmacokinetic (PK) interactions and to potentiate immunosuppressive efficacy in a highly stringent rat lung allograft model. Methods: Lew recipients of BN lungs were treated orally daily with: A) RAD 2.5 mg/kg (n=9), B) Neoral 7.5 mg/kg (n=8), C) RAD 2.5 mg/kg+Neoral 7.5 mg/kg simultaneously(n=8) or D) RAD 2.5 mg/kg+Neoral 7.5 mg/kg (n=6) staggered 6 hrs apart. Rats were assessed by daily weights, chest X-rays, drug trough levels (LC/MS) and blinded scoring of graft histology at sacrifice (d 21). Results: X-rays were completely opacified in all grafts of controls and the RAD monotherapy group on d 7 and 14, respectively and on d 21 were mildly opacified (Neoral monotherapy) and completely clear (both RAD+Neoral groups). Combined treatment dramatically reduced histologic rejection compared to treatment with either drug alone (Fig). Simultaneous treatment caused toxicity (poor grooming, lethargy) and significantly higher day 14 trough RAD and CsA levels (49±5/638±106 ng/ml; p<0.04) than for the staggered group (30±1/378±20 ng/ml) in which all animals were clinically normal. RAD and CsA day 14 trough levels in the staggered group were the same or lower than trough levels in animals treated with either drug alone (RAD 27±3 /Neoral 815±67 ng/ml).Conclusions: 1) Administration of RAD+Neoral suppresses lung rejection more effectively than treatment with either drug alone, 2) RAD and CsA trough levels do not differ between monotherapy and staggered,combination therapy, suggesting that potentiation of efficacy due to coadministration is due to pharmacologic, rather than PK, interactions.3) Staggered, rather than simultaneous, administration of RAD+Neoral,avoids the PK interactions that cause elevated drug blood levels and toxicity. Thus, we have potentiated efficacy and reduced toxicity by staggering administration of RAD and Neoral.

BENEFITS OF PREEMPTIVE DOSE REDUCTION FOR SANDIMMUNE TO NEORAL CONVERSION IN STABLE RENAL TRANSPLANT RECIPIENTS

241 We previously reported a high incidence of nephrotoxicity in stable renal transplant patients converted from Sandimmune to Neoral using a 1:1 dosing ratio. Serum creatinine concentration (sCr) transiently increased to more than 30% above baseline in 68 of 141 (48%) consecutive patients. Moreover, 74% of patients required a reduction in dose after conversion. This experience prompted the current study in which 23 stable patients were converted from Sandimmune to Neoral using a preemptive 20% dose reduction (group 1). Outcomes of patients in group 1 were compared to those of 23 patients converted using a 1:1 dosing ratio and matched for age, baseline sCr, and baseline trough CsA levels (group 2). Baseline Sandimmune dose was 4.3±1.6 mg/kg/d in group 1 and 4.6±1.4 mg/kg/d in group 2 (p=NS). Neoral dose was reduced on the basis of an elevated sCr (>15% above baseline) or a lesser elevation associated with a CsA level >200 ug/L. In each group, baseline sCr and trough CsA levels were compared to those obtained 4 days, 11 days, and at last follow-up after conversion: TableThe percent change in peak sCr after conversion was 12±20% in group 1 and 37±41% in group 2 (p<0.04). In group 1, 3 of 23 patients required additional Neoral dose reductions while 15 of 23 patients in group 2 required dose reductions (p=0.001). However, at last follow-up, the dose reductions in each group were not different (-21±11%, group 1 vs-19±18%, group 2; p=NS). Conclusion: Compared to conversion using a 1:1 dosing ratio, conversion from Sandimmune to Neoral using a preemptive 20% dose reduction results in less short-term nephrotoxicity and comparable dose reductions. Preemptive dose reduction may allow Sandimmune to Neoral conversion with less frequent laboratory monitoring.

COADMINISTRATION OF MICROEMULSION CYCLOSPORINE AND THE NOVEL LEFLUNOMIDE MALONONITRILAMIDE ANALOG, HR1279, MORE EFFECTIVELY SUPPRESSES RAT LUNG ALLOGRAFT REJECTION THAN MICROEMULSION CYCLOSPORINE PLUS LEFLUNOMIDE

522 HMR 1279 (279) is one of two new malononitrilamides being developed as alternatives to leflunomide (LFM) as immunosuppressants for transplantation. This study evaluated the relative immunosuppressive efficacies of daily PO(gavage) treatment with cyclosporine (Neoral), LFM or 279 monotherapy and combined therapy with Neoral plus either LFM or 279 in an aggressively rejecting rat lung allograft model. Method: 36 Lew recipients of unilateral BN lungs were monitored by daily weights, chest radiographs, drug trough levels (HPLC for 279 and A77 1726, the active metabolite of LFM) and LC/MS for CsA until d 21 when grafts were removed for blinded histologic grading by ISHLT criteria. Groups (n = 6 for all groups) were: untreated controls, Neoral (7.5 mg/kg), 279 (10 mg/kg) or LFM (10 mg/kg); the same doses were then used in combination therapy regimens: Neoral + LFM and Neoral + 279.Results: Other than 1 death, all recipients remained clinically healthy with an average 4-6% percent change in body wt. Chest radiographs showed moderate to complete opacification of grafts in control rats on d 7 and by d 14 in LFM and 279 monotherapy groups, but were clear on d 21 in the remaining groups. Histologic scoring (fig) showed moderate to severe rejection in grafts from control rats and rats treated with 279 or LFM. The efficacy of Neoral alone was not improved by its combination with LFM even though combination therapy significantly (p<0.001) increased trough serum levels of A77 1726 (1.9±0.7 ug/ml)compared to levels during LFM monotherapy (0.2±0.2 ug/ml). Neoral + 279, however, suppressed histologic rejection substantially more than any other treatment regimen. CsA levels were not affected by coadministration of Neoral with LFM or 279. Conclusions: 1) Neoral, LFM and 279 monotherapy suppress lung graft rejection much less effectively than BN to Lew heart graft rejection based on previous work using identical drug regimens, 2) Lung graft rejection is most effectively suppressed by Neoral + 279, 3) In contrast, the efficacy of Neoral is not improved by coadministration of LFM, 4) This the first study to show 279 is more immunosuppressive than LFM; the reasons for the differences in efficacies between the analog and parent drug are being investigated.

THE EFFECT OF DIFFERENT IMMUNOSUPPRESSION THERAPY ON ERYTHROPOIETIN PRODUCTION IN RENAL TRANSPLANTATION

51 Anemia in chronic renal failure is due to erythropoietin (Epo) deficiency that is expected to normalize after successful kidney transplantation. We previously studied 40 children with excellent graft function for 12 months post kidney transplantation and found a mean hematocrit (Hct) -2 SD from the mean for age and sex. Human hepatoma (Hep3B) cells were chosen as a model to study the effect of immunosuppression therapy on Epo production. Cyclosporine(CSA), Azathioprine (AZA), FK506, and Mycophenolate Mofetil (MPM), under 21% O2 and 1% O2 (hypoxic) conditions were tested. Epo levels were measured in mU/mg protein ± SEM and compared to baseline conditions. Both CSA(10μg/mL) and AZA (40μg/mL) inhibited Epo production under 21% O2,(p<.03, p<.002) and hypoxic conditions (p<.02, p<.05) respectively. Increased concentration of CSA and AZA (100μg/mL) resulted in greater inhibition. FK506 (.0001 -1μM) and MPM (.01-10μM) did not inhibit Epo production. CSA added to high dose AZA or MPM depressed Epo production further than either agents alone in 21% O2 (p<.002 and p<.007) and 1% O2 (p<.004 and p<.0001). We then measured Hct%, plasma Epo, and ferritin, in 4 pediatric renal transplant recipients, ages 4-8 years, 3 were on CSA and one on FK506 at 1, 2, 3, 6, 9 months post transplantation. Simultaneous blood trough levels for CSA were done (whole blood TDx), and for FK506 (whole blood MEIA). No acute rejection episodes or blood transfusions occurred in any patient. Mean Hct% for all patients pre transplant, and then at 1, 3, 6, 9 months post transplantation were 36%, 33%, 38%, 37%, and 38%. All patients had adequate ferritin levels (range of 30-167 ng/ml) in the first 6 months post transplantation. Mean serum creatinine at 6 months was 0.65±.2 mg/dl. Epo levels were compared to each Hct value according to a Reference Regression Graph. An inverse linear relationship between log Epo levels and Hct(R2 = 0.341, p=.0002) was found that was less steep than for healthy adults, and similar to patients with renal failure. No correlation was found between CSA or FK506 levels and Hct. Conclusion: CSA and AZA suppress in-vitro Epo production while FK506 and MPM appear to have no effect. The Epo levels produced after transplantation is inappropriately low compared to Hct. We speculate that the mild anemia in children post renal transplantation is due to inhibition of renal interstitial Epo production related to immunosuppression therapy, specifically CSA. The effect of FK506 and MFM on Epo production in-vivo needs further study.

A RANDOMISED STUDY OF CONVENTIONAL VS LOW DOSE CYCLOSPORINE IN RENAL TRANSPLANT RECIPIENTS TREATED WITH CYCLOSPORINE, MYCOPHENOLATE MOFETIL AND PREDNISONE.

418 The addition of mycophenolate mofetil (MMF) to cyclosporine (CsA) and prednisone (Pred) has reduced the rate of biopsy-proven acute rejections after renal transplantation (RTx) by nearly 50%. It is unknown whether this result can also be obtained with a reduced dose of CsA, in order to minimize the incidence of CsA-related side effects. Methods: In this multicentre study, recipients of a first or second renal allograft were randomised to treatment with MMF 1000 mg bid, Pred, and either the conventional (“high”) or a low dose of CsA during the first three months after RTx. Target whole blood trough levels of CsA were 300 and 150 ng/ml, respectively. Data regarding the first three months after RTx were analysed. Results: 110 patients were included: 51 patients received the high dose and 59 the low dose of CsA. Graft failure or patient death occurred in 8/51 patients (16%) in the high CsA group and in 4/59 patients (7%) in the low CsA group (p=0.22). Acute rejection occurred in 9/51 patients (18%) in the high CsA group and in 17/59 patients (29%) in the low CsA group (p=0.19). First line treatment of acute rejections consisted of i.v. methylprednisolone. The use of anti thymocyte globulin was necessary in 3/9 cases (33%) in the high CsA group and in 6/17 cases (36%) in the low CsA group. The incidence of delayed graft function was similar in both groups (9/51 vs 8/59; NS). Serum creatinine at 3 months did not differ between the high and low CsA group: 144± 44 μmol/l vs 130 ± 46 μmol/l (p=0.14). The incidence of CMV disease was similar in both groups (11/51 and 11/59 patients, respectively). Conclusion: These results suggest that the addition of MMF to a combination of CsA and Pred allows the use of a lower than conventional dose of CsA after renal transplantation.

LOW-DOSE CYCLOSPORINE AND MYCOPHENOLATE MOFETIL IN RENAL ALLOGRAFT RECIPIENTS WITH SUBOPTIMAL RENAL FUNCTION.

417 Cyclosporine (CsA) nephrotoxicity can be identified by functional changes and chronic renal damage. CsA-associated renal fibrosis has been related to the overproduction of TGF-ß, a fibrogenic cytokine. CsA dose reduction may improve renal function but may increase the risk for rejection. We have studied the impact of CsA dose reduction in association with mycophenolate mofetil (MMF) on renal function and TGF-ß production in long-term (> 12 months after transplantation) renal allograft recipients with suspected CsA nephrotoxicity (serum creatinine 140-300 μmol/l, proteinuria < 1 g/day). In these patients MMF was introduced at 2 g/day and CsA dose was reduced to reach CsA whole blood levels between 40-60 ng/ml in one month. Before and 6 months after the introduction of MMF, CsA dose and levels, renal function parameters, and TGF-ß in platelet-free plasma, were evaluated. Eighteen patients were entered in this study, and 2 of them were withdrawn because of MMF intolerance. The results obtained in the remaining 16 cases (10 M/6F, mean age 44 y) are summarized in the next table:CsA levels correlated with TGF-ß values (r= 0.536, p= 0.0016). No rejection episodes occurred in these patients. These results show that MMF introduction allows CsA dose reduction which ameliorates renal function and reduces TGF-ß production.

Cyclosporine metabolite cross-reactivity in different cyclosporine assays.

There is a controversy regarding the role of cyclosporine (CsA) metabolites in both immunosuppression and toxicity, and measurement of the parent drug is commonly recommended. High performance liquid chromatography (HPLC) is the method commonly used for specific measurement of the parent drug, but is very time consuming. Antibody techniques are available but vary in specificity. Mixed lymphocyte culture assay (MLC) is a functional bioassay for the measurement of CsA which measures both parent drug and active metabolites. Because it is time consuming and labor intensive, it is not practical to use the MLC to monitor patient's CsA levels. The objective of this study is to evaluate the degree of cross-reactivity or interference among two different CsA immunoassays [(Immunoassay: CYCLO-Trac-RIA, Monoclonal-TDX; and two radioreceptor assays (RRA) (52 kDa immunophilin and cyclophilin)] with seven cyclosporine metabolites (AM19, AM1c9, AM4n9, AM1, AM9, AM1c, AM4n). The results are compared with a previously published MLC assay for the same metabolites. 500 ng/mL of each of the CsA metabolites was assayed in spiked blood samples with both RRA using 52 kDa immunophilin and commercial cyclophilin and two commonly used commercial immunoassay procedures. The results were compared to those obtained with the previously published MLC assay. The CYCLO-Trac-radioimmunoassay showed minimal cross-reactivity with all of the seven CsA metabolites tested and is more specific to parent CsA than the current Abbott monoclonal procedure for the measurement of CsA. However the cross-reactivity of the seven metabolites using the Abbott monoclonal assay matched closely with their pharmacological potency as measured in the MLC assay. The RRAs showed greater cross-reactivity for most of the CsA metabolites tested than that found in the immunoassay procedures.

Quantitation of immunosuppression by flow cytometric measurement of the capacity of T cells for interleukin-2 production.

Methods to quantitate the effects of immunosuppressive drugs on immune reactivity might be helpful for monitoring immunosuppressive treatment. Cyclosporine (CsA) inhibits the induction of cytokine synthesis in T cells, and measurement of interleukin (IL)-2 production might constitute a parameter of this drug's effect. We determined the percentages of CD4+ and CD8+ lymphocytes producing IL-2 upon stimulation by phorbol myristate acetate and calcium ionophore in whole blood culture, using immunostaining of intracytoplasmatic and membrane markers, followed by multiparameter flow cytometry. A total of 38 clinically stable transplant patients on various immunosuppressive protocols were studied. The percentage of CD4+ T cells producing IL-2 was strongly reduced in patients compared with healthy controls (23% [range, 3-68%] vs. 59.0% [range, 41-70%]; P=0.000035). The percentage of CD4+ T cells producing IL-2 was negatively correlated with the CsA level (Rc=-0.0821, P=0.00002297) but not with prednisolone or azathioprine doses. Fewer CD8+ T cells produced IL-2 in transplant patients compared with controls, but the difference failed to reach statistical significance. The percentage of CD8+ T cells capable of producing IL-2 was inversely correlated to CsA levels (Rc=-0.0375, P=0.0011). These data suggest that the functional effects of CsA in transplant recipients can be quantitatively determined and that the capacity of CD4+ T cells to produce IL-2 upon stimulation constitutes a functional parameter of CsA effects on the immune system. Prospective studies are required to determine whether this method is useful for clinical monitoring.

Steroid withdrawal is safe and beneficial in stable cyclosporine-treated liver transplant patients

Background: In the immunosuppression of orthotopic liver transplant recipients, steroids are used despite their unspecific action and long-term side effects. Few studies have been carried out on steroid withdrawal and many aspects remain to be elucidated. Methods: A prospective study was performed to analyse the effect of steroid withdrawal on 86 patients with stable graft function, more than 1 year after orthotopic liver transplant. Thirty patients had chronic hepatitis in the graft. Seventy-two continued with cyclosporine (CsA) and 14 with CsA-azathioprine (AZA) therapy. The follow-up was 23.2±8.1 months (range (12–52 months). A paired t-test was used for statistical analysis. Results: No acute or chronic rejection occurred, and steroids were not reinstituted. There were no changes in serum transaminase levels, but bilirubin levels decreased ( p<0.01). At the end of the follow-up, we found improvements in blood pressure in hypertensive patients (systolic 156.1±8.4 mmHg vs. 139.4±8.7 mmHg, p<0.001); body weight (72±13.5 kg vs. 70.8±13 kg, p<0.05); serum cholesterol (211.3±42 mg/dl vs. 191.6±43.5 mg/dl, p<0.001) and bone mineral density in lumbar spine (0.823±0.13 g/cm 2 vs. 0.893±0.135 g/cm 2, p<0.001). Four of ten diabetic patients were no longer insulin-dependent and insulin requirements decreased in the remaining six. No significant biochemical changes were found in patients with hepatitis in the graft, and we found an improvement in inflammatory activity in the nine biopsed patients. Conclusions: Steroid withdrawal with CsA monotherapy is feasible, safe and beneficial in patients who have stable liver graft function 1 year after orthotopic liver transplant. We consider that AZA therapy is not necessary unless drastic reduction of CsA levels is required because of renal dysfunction.

Breast-feeding during treatment with cyclosporine.

Mothers treated with cyclosporine (CsA) have previously not been allowed to breast-feed due to the reported accumulation of the drug in breast milk. The purpose of this study was to evaluate the consequences of allowing breast-feeding. Seven infants were breast-fed by mothers who had undergone kidney transplantation alone (n=5) or simultaneous kidney and pancreas transplants (n=2). In addition to CsA, all mothers received prednisolone at 5-7.5 mg/day and six mothers received azathioprine at 50-100 mg. CsA concentration was measured in the whole blood of mothers and babies and in breast milk. Serum creatinine was measured in babies 1 week after birth and after 4-12 months of breast-feeding. Blood CsA levels ranged from 55 to 130 ng/ml in mothers (12-hr trough), 50 to 227 ng/ml in breast milk (mean for each woman), and was below the detection limit of 30 ng/ml in all infants. Breast milk concentration ranged from 87 to 440 ng/ml in 16 samples obtained at various time points from one mother. Infants' serum creatinine ranged from 25 to 54 micromol/L at 1 week after birth and 23-52 micromol/L after breast-feeding. All babies thrived. Breast-fed infants of mothers treated with CsA received less than 300 microg per day of CsA and absorbed undetectable amounts. There were no demonstrable nephrotoxic effects or other side effects. Thus, women with kidney transplants could be allowed to breast-feed.

Effect of early cyclosporine levels on kidney allograft rejection

Acute rejection is the greatest risk factor for development of biopsy‐proven chronic rejection and late kidney allograft loss. We previously noted that low cyclosporine (CSA) levels were a risk factor for early acute rejection in pediatric recipients (1). In our current study, we used logistic regression to identify risk factors for acute rejection in 726 adult kidney transplant recipients on triple therapy (prednisone, azathioprine, CSA). Variables considered for logistic regression analysis were donor organ source (cadaver vs. living), degree of HLA mismatch (1 to 6 vs. O antigen mismatch), transplant number (primary vs. retransplant), CsA levels (&lt; 125 vs. ≥ 125 ng/ml, &lt; 150 ng/ml vs. ≥ 150 ng/ml, and &lt; 175 vs. ≥ 175 ng/ml), and acute rejection episodes (0 vs. ≥ 1). Of 726 recipients, 401 (55%) received cadaver kidneys; 325 (45%), living related. Overall, 572 (79%) had a primary transplant; 154 (21%), a retransplant. The vast majority of acute rejection episodes occurred within the first 2 months posttransplant; 68% of recipients had no acute rejection episodes by 2 months and 58% had none by 60 months posttransplant.Logistic regression analysis revealed that a cadaver donor kidney (vs. living) (p = 0.004), a 1 to 6 antigen mismatch (vs. 0 mismatch) (p = 0.001), and CsA levels &lt; 150 ng/ml (vs. ≥ 150 ng/ml) correlated with biopsy‐proven acute rejection. The correlation for CsA levels &lt; 150 ng/ml (vs. ≥ 150 ng/ml) held true for levels at 1 wk (p &lt; 0.05), 1 month (p = 0.0001), 2 months (p = 0.01), and 3 months (p = 0.02) posttransplant. Similar correlation was found for CsA levels &lt; 125 ng/ml (vs. 2 125 ng/ml) and &lt; 175 ng/ ml (vs. 175 ng/ml).Comparative analyses were made (by Chi‐square) of acute and chronic rejection rates when recipients were divided into 3 groups by CsA level (&lt;125 ng/ml, 125 to &lt; 150 ng/ml, and ≥ 150 ng/ml). At each time point (1) wk, 2 wk, 1 month, 2 months, 3 months), CsA levels &lt; 125 ng/ml (vs. ≥ 125 to &lt; 150 ng/ml and ≥ 150 ng/ml) were associated with the greatest increased risk of acute rejection ‐ for both cadaver and living related recipients (all p&lt;0.05). CsA levels &lt; 125 ng/ml at each time point (1 wk, 2 wk, 1 month, 2 months, 3 months) were also associated with a significantly increased risk of chronic rejection (all p&lt;0.001). The incidence of both acute and chronic rejection was reduced in the group with CsA levels ≥ 125 to &lt; 150 ng/ml and further reduced in the ≥ 150 ng/ml group. Our data indicate that maintaining CsA levels ≥150 ng/ml as part of triple therapy reduces the incidence of both acute and chronic rejection. Because chronic rejection is the leading cause of late allograft loss, maintaining adequate CsA levels should improve long‐term graft survival. Based on this analysis, we have modified our own immunosuppressive regimens to achieve higher CsA levels earlier posttransplant.

Calcineurin activity in children with renal transplants receiving cyclosporine.

The major immunosuppressive effect of cyclosporine is through the inhibition of calcineurin, an enzyme important in the activation of T lymphocytes. In children, neither calcineurin activity nor its inhibition by cyclosporine (CsA) has been investigated. Calcineurin activity, was measured in stable pediatric renal transplant patients, with healthy children used as controls. Whole blood CsA concentrations were measured by monoclonal radioimmunoassay. Simultaneous calcineurin and CsA levels were measured before and 1, 2, 3.5, 5, and 12 hr after their routine morning CsA dose. Calcineurin activity was approximately 50% inhibited at trough blood concentrations (148 microg/L); moreover, inhibition increased as CsA concentrations rose and declined as concentrations fell. Maximum calcineurin inhibition was about 70% at concentrations of about 431 microg/L. Linear regression analysis revealed a significant correlation between mean CsA blood concentration and the mean degree of inhibition of calcineurin activity (P=0.005, one-tailed). We conclude that inhibition of calcineurin activity by CsA in pediatric renal transplant recipients correlates with CsA blood concentrations.

Intrasplenic hepatocyte allotransplantation in dalmation dogs with and without cyclosporine immunosuppression.

Hepatocyte allotransplantation has been performed successfully in several small animal models for the amelioration of inborn metabolic errors. Before a human clinical trial of hepatocyte allotransplantation can be attempted, preliminary experience in a large animal model is needed. We transplanted isolated mongrel hepatocytes into the spleen of dalmatians in the attempt to cure their inborn error of uric acid metabolism. Of 10 dalmatian recipients, two that received 9-10 x 10(9) mongrel hepatocytes died early after surgery of acute portal hypertension and hemorrhage. The eight long-term survivors received 5-6 x 10(9) hepatocytes and were randomized either to no treatment or to oral cyclosporine (CsA). Levels of CsA were adjusted to maintain trough levels between 400 and 800 ng/ml. In the four nonimmunosuppressed dalmatians, a reproducible average reduction in urinary uric acid excretion (UUAEx) of 23.7% was achieved; values returned to baseline within 14 days. In the CsA-immunosuppressed dalmatians, the average decline in UUAEx was 30%. The partial correction of the metabolic defect persisted for an average of 25 days in three immunosuppressed dogs, whereas in one dog, the partial correction lasted for over 90 days. No change in UUAEx was observed in two dalmatians that underwent sham laparotomy and intrasplenic injection of saline solution; CsA given alone to dalmatians did not modify UUAEx. We conclude that the dalmatian dog is a valuable large animal model for studies of the role of hepatocyte transplantation in the cure of inborn hepatic metabolic errors.

BUDESONIDE, A LOCALLY ACTING STEROID, PREVENTS GRAFT REJECTION IN A RAT MODEL OF INTESTINAL TRANSPLANTATION1

The requirement for potent systemic immunosuppression to prevent intestinal graft rejection has resulted in high rates of infection and post-transplant lymphoproliferative disease. Budesonide (BUD) is a locally acting steroid that is almost completely metabolized during its first pass through the intestinal wall and liver. The present study examined whether BUD could prevent small bowel allograft rejection without causing the adverse systemic effects associated with prednisolone. Orthotopic Brown Norway to Lewis rat small bowel allotransplants were randomly assigned to treatment with low-dose BUD (0.1 mg/kg/day, p.o.) and high-dose BUD (1.0 mg/kg/day, p.o.) with and without cyclosporine (CsA; 2 mg/kg/day s.c.). The following parameters were assessed: allograft survival, recipient plasma adrenocorticotrophic hormone (ACTH) levels, recipient adrenal, splenic and thymic weights, recipient CsA levels, and graft histopathology. Low- and high-dose BUD alone did not prolong graft survival compared with the untreated group (9.1+/-0.4 days vs. 11+/-0.8 days vs. 9.7+/-0.4 days, respectively). However, when low-dose BUD and high-dose BUD were given in combination with CsA, the mean graft survival times were prolonged to 27.6+/-5.3 and 36.6+/-8.0 days, respectively (P<0.01). ACTH levels, adrenal weights, and thymic weights were not significantly different in the treatment and control groups receiving intestinal transplants. BUD enhances the immunosuppressive effects of CsA and prolongs small bowel allograft survival in rats without inhibiting normal ACTH release. These data suggest that BUD may be a useful immunosuppressive agent for clinical intestinal transplantation.

Neoral cyclosporine (CSA) kinetics in children treated with diltiazem. 1686

The new microemulsion formulation of CSA (Neoral) has supplanted conventional Sandimmune, with little formal evaluation of Neoral kinetics in the pediatric population. CSA kinetics and area under the curve(AUC) were measured in 19 children receiving both Neoral and diltiazem following either renal (n=13) or hepatic (n=6) transplantation. The following parameters (mean + 1SD) were observed in 15 children taking Neoral on a bid schedule: dose (mg/kg/d): 5.8+2.1; trough (ng/ml): 204+76; daily AUC (h× ng/ml): 11045+2398; Tmax (hrs): 1.37+0.48; Cmax (ng/ml): 1227+263. Although the correlation between trough levels and AUC is said to be improved with Neoral in adults, this was not the case in our experience (r=0.46, p=0.08). The best correlation was noted with the 2 hr CSA level (r=0.84, p< 0.0001) or the 6 hour level (r=0.77, p<0.001). In 9 children (4 renal, 5 hepatic), kinetic studies and iothalamate clearance (GFR) were also available on both Sandimmune and Neoral following dose adjustment to maintain a constant trough. Conversion from Sandimmune to Neoral was associated with an increase in AUC and Cmax despite maintenance of constant trough levels. There was also a trend toward a lower dose, more rapid peak, and a lower GFR (p=0.06).

Diagnostic contribution of renal allograft biopsies at various intervals after transplantation.

Renal allograft biopsy is the accepted gold standard for investigating episodes of graft dysfunction in the early posttransplant period. The situation is less clear in late transplant biopsies. Later renal biopsies performed for graft dysfunction or as part of a routine investigative protocol have not been subjected to detailed critical evaluation. Two hundred sixty-three consecutive renal allograft biopsies in a single center were evaluated. They were arbitrarily divided into three groups based on interval after transplantation: group 1, up to 3 months (n=117); group 2, 4-12 months (n=60); and group 3, greater than 12 months after transplantation (n=86). There were no significant differences in demographic factors among the groups. The mean interval after transplantation was 0.8+/-0.1 months in group 1, 6.1+/-0.3 months in group 2, and 40.1+/-3.4 months in group 3. There were six principal diagnostic categories: acute rejection (AR), chronic rejection (CR), cyclosporine (CsA) nephrotoxicity, acute tubular necrosis (ATN), normal, and others. A statistically significant decrease in the frequency of AR (P<0.001) was seen in group 3 (3%) compared with groups 1 (43%) and 2 (37%). In contrast, the frequency of CR was significantly higher (P<0.001) in group 3 (71%) compared with groups 1 (0) and 2 (10%). ATN was seen almost exclusively in group 1. All but one of the 37 patients with ATN were in this group. CsA nephrotoxicity remained an important cause of graft dysfunction in all three groups, with no significant difference in incidence among the three groups. The differences between groups with other histological types were not significant. Patient management was changed based on the biopsy report in 84 patients in group 1 (72%), 45 patients in group 2 (75%), and only 16 patients in group 3 (19%) (P<0.001). In only seven patients in group 3 did the change in management result in a significant change in serum creatinine. All of these seven patients had CsA nephrotoxicity on biopsy and also had a significantly higher level of CsA compared with those with AR or CR. Thus, the diagnosis might have been possible without the need for biopsy. We conclude that late renal allograft biopsies are only rarely helpful in patient management and as such should be an investigation of last resort.