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Drug Monitoring and Toxicology |
1 Servei Immunologia,
2 Servei de Toxicologia, and
3 Unitat de Trasplantament Renal, IDIBAPS, Hospital Clínic de Barcelona, 08036 Barcelona, Spain.
aAddress correspondence to this author at: Hospital Clínic Servei Immunologia, C/Villarroel 170, 08036 Barcelona, Spain. Fax 34-934518038; e-mail jmarto{at}clinic.ub.es.
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Abstract |
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Methods: We treated 65 stable renal transplant recipients with cyclosporin A (CsA; n = 16), tacrolimus (TRL; n = 10); CsA + mycophenolate mofetil (MMF; n = 14); TRL + MMF (n = 13), and MMF (n = 12). Twelve nontreated healthy controls were also included. Calcineurin activity (CNA) in peripheral blood mononuclear cells was measured using 32P-labeled peptide. Interleukin-2 (IL-2) and interferon-
production in phytohemagglutinin-activated whole blood were measured at 0 and 2 h postdose. The areas under the curves, cmin, cmax, and concentration at 2 h (c2 h) were also measured.
Results: We found no differences in CNA between groups receiving CNIs alone or combined with MMF [median (25th–75th percentiles)]: CsA2 h, 3.87 (3.00–6.85)% alkaline phosphatase (AP); CsA+MMF2 h, 3.90 (1.78–5.19)% AP; TRL2 h, 5.68 (3.02–16.00)% AP; TRL+MMF2 h, 11.80 (4.05–14.63)% AP. In vitro IL-2 production was significantly lower in the groups receiving combined therapy than in groups receiving CNIs alone [median (25th–75th percentiles)]: CsA2 h, 276.52 (190.41–385.25) ng/L; CsA+MMF2 h, 166.48 (81.06–377.01) ng/L (P <0.001); TRL2 h, 249.34 (127.48–363.50) ng/L; TRL+ MMF2 h, 122.13 (51.02–180.00) ng/L (P <0.001). The correlations (r) between c2 h and CNA 2 h postdose were as follows: CsA, r = -0.74; CsA+MMF, r = -0.84; TRL, r = -0.70; TRL+ MMF, r = -0.70 (P <0.001 in all cases).
Conclusions: The measurement of CNA may be of help in following the effect on the immune system of CNI treatments, even in combined therapies, but does not reflect the additional effect of MMF. In contrast, IL-2 in vitro production reflects the effect of both MMF and CNIs.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The measurement of blood concentrations of immunosuppressants provides only an indirect evaluation of the degree of immunosuppression attained in an individual patient (1)(2) When combined treatments are used, the biological impact may be higher than that predicted by the individual blood concentrations of each immunosuppressant. Thus the measurement of several markers has been proposed to assess the impact of immunosuppressants on the immune systems of individual patients. Various approaches have been proposed for the pharmacodynamic (PD) monitoring of immunosuppressants: One is evaluation of the activity of the specific target enzyme, such as calcineurin activity (CNA) for cyclosporin A (CsA) and tacrolimus (TRL) (3)(4)(5), inosine monophosphate dehydrogenase for MMF (6)(7)(8), and P70S6 kinase activity for sirolimus (1)(9). A second approach is the evaluation of an intermediate step in the action mechanism: for example, the measurement of interleukin-2 (IL-2) and interferon- (IFN-) production in vitro in whole blood for CsA and TRL (10), and DNA duplication for MMF and sirolimus (7)(11). A third possibility is the measurement of collateral markers modified by the presence of the immunosuppressant, such as the T-lymphocyte surface antigens CD25, CD71, and CD154 (12)(13).
CsA and TRL are widely used to prevent rejection in allotransplantation. Both drugs have the same target: the serine/threonine phosphatase calcineurin (CN), or PP2B. One important substrate of this phosphatase is the nuclear factor of activated T cells. Nuclear factor of activated T cells remains in the cytoplasm when it is inactive. It is activated by dephosphorylation effected by CN and migrates to the nucleus, where it participates in the induction of the transcription of genes necessary for lymphocyte expansion, such as IL-2, IFN-, IL-4, IL-3, and tumor necrosis factor-
(14). To inhibit CNA, CsA and TRL must bind to the immunophilins present in the cytoplasm of T lymphocytes: cyclophilins in the case of CsA and FK-binding proteins (FKBPs) in the case of TRL. CsA–cyclophilin (CsA–cyclophilin A) and TRL–FKBP (TRL–FKBP12) complexes inhibit 
80–90% of CNA (15)(16)(17).
To our knowledge, no data on the correlation between CNA and IL-2 or IFN- production have been reported in stable renal patients receiving CsA or TRL alone or in combined therapy with MMF.
The aims of this study were (a) to determine the correlation between the pharmacokinetic (PK) parameters [cmin, cmax, c2 h, and area under the curve (AUC)] for CsA and TRL and the different PD indices proposed (CNA and IL-2 and IFN- production), and (b) to assess the importance of combined therapies on the measurement of CNA and IL-2 and IFN- production. Establishing additional PD indices to measure the biological impact of immunosuppressants can be of help in the introduction of new immunosuppressant combinations, in the introduction of lower-than-standard doses, and in the study of individual variability in certain special cases.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We recruited 12 healthy individuals [NHC; mean age 43 (8) years] as a control group for basal determinations.
reagents
Protein kinase (3':5'-cyclic AMP-dependent, from bovine heart); okadaic acid; the protease inhibitors aprotinin, leupeptin, and soybean trypsin; phenylmethylsulfonyl fluoride; dithiothreitol (DTT), MES; Tris; phytohemagglutinin (PHA); and trifluoroacetic acid (TFA) were purchased from Sigma. Acetonitrile was provided by Scharlau. CN substrate was obtained from Neosystem S.A. (Groupe SNPE) and [-32P]ATP (10 Ci/L) was purchased from Nuclear Ibérica S.A. C18 solid-phase extraction columns (Sep-Pak) were from Supelco®. AG50W-X8, 100–200 mesh, cation-exchange resin was obtained from Bio-Rad Laboratories. Alkaline phosphatase (AP; 20 U/µL) was from Boehringer Mannheim. OptiPhase "HiSafe" 2 was from Wallac Scintillation Products. CsA and TRL were kindly supplied by Novartis Farmacéutica S.A. (Barcelona, Spain) and Fujisawa Pharmaceutical Co, Ltd.(Osaka, Japan), respectively. Trichloroacetic acid and K2HPO4 were provided by Merck. Human IL-2 and human IFN- immunoassays were obtained from Immunotech.
cell preparation
Human peripheral blood mononuclear cells (PBMCs) were obtained from the mononuclear cell layer of the Ficoll-Hypaque gradient. The remaining red blood cells were lysed during a short incubation period (10 s) in water followed by addition of an equal volume of phosphate-buffered saline (2x concentrate). Washed and pelleted PBMCs (6 x 106) were lysed with 0.3 mL of hypotonic lysis buffer containing protease inhibitors [50 mmol/L Tris (pH 7.5), 0.1 mmol/L EGTA, 1 mmol/L EDTA, 0.5 mmol/L DTT, 50 mg/L phenylmethylsulfonyl fluoride, 50 mg/L soybean trypsin, 5 mg/L leupeptin, and 5 mg/L aprotinin]. Lysis was facilitated by three rounds of freezing in liquid nitrogen followed by thawing at 30 °C. Cellular debris was sedimented by centrifugation at 4 °C for 10 min at 12 000g, and the clear supernatant was frozen and stored for at most 2 weeks in liquid nitrogen without significant loss of CN activity (18).
phosphorylation of synthetic peptide
A 19-amino-acid peptide derived from cAMP-dependent kinase regulatory subunit type II was phosphorylated in vitro and used as a substrate to measure CNA. The 19-amino-acid peptide (19mer) sequence was Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Ala-Ala-Glu. The lyophilized peptide was dissolved in water to a concentration of 3.30 mmol/L. Phosphorylation of the serine residue with [-32P] ATP was performed with the catalytic subunit of cAMP-dependent kinase. The kinase reaction contained 50 µL of buffer A [40 mmol/L MES (pH 6.5), 0.40 mmol/L EGTA, 0.80 mmol/L EDTA, 4 mmol/L MgCl2, 0.10 mmol/L CaCl2, and 0.10 g/L bovine serum albumin], 12 µL of [-32P] ATP (10 Ci/L), 9 µL of 3.30 mmol/L peptide, 19 µL of H2O, and 10 µL of 160 mg/L protein kinase. After the reaction mixture had incubated for 1 h at 30 °C, we added 900 µL of H2O.
The phosphorylated peptide was purified by Sep-Pak C18 chromatography. Columns were prepared using syringes to apply 3 mL of 300 mL/L acetonitrile in 1 g/L TFA followed by 5 mL of 1.0 g/L TFA. The contents of the kinase reaction were then slowly applied to the column. The column was washed with 400 mL of TFA to remove unincorporated ATP. The peptide was then eluted by addition of five 0.5-mL volumes of 300 mL/L acetonitrile in 1.0 g/L TFA. The radiolabeled peptide fractions were pooled and evaporated under argon gas, resuspended with 2 mL of buffer 1 [20 mmol/L Tris-HCl (pH 8), 100 mmol/L NaCl, 6 mmol/L MgCl2, 0.10 mmol/L CaCl2, 0.50 mmol/L DTT, and 0.10 g/L bovine serum albumin], and stored at -20 °C (18).
The interassay CV for peptide labeling was 23% and was assessed by phosphorylating the synthetic peptide on different days with the same batch of [-32P]ATP and protein kinase. The intraassay CV of the phosphorylation assay was 5.6% and was assessed by performing five replicates of the phosphorylation assay on the same day.
phosphatase assay (cna measurement)
CNA was measured in treated patients and in the NHC group in the morning predose and 2 h postdose.
Hypotonic lysates of PBMCs were evaluated for their ability to dephosphorylate a 32P-serine-labeled 19-amino-acid peptide substrate (19mer) in the presence of okadaic acid, a phosphatase type 1 and 2A inhibitor, as described previously by Fruman et al. (18). Background phosphatase 2C activity (CsA- and okadaic acid-resistant activity) was determined and subtracted from each sample, with the assay performed in the presence and absence of excess added CsA or TRL. The remaining phosphatase activity was taken as CNA (PP2B).
Assays were performed in a final volume of 60 µL, in four different tubes. In all tubes we added 20 µL of PBMC lysates and 20 µL of 32P-labeled phosphopeptide (final concentration, 5 µmol/L) as substrate. In addition, in tube 1 we added 20 µL of buffer 1, in tube 2 we added 20 µL of buffer 2 (buffer 1 containing 500 nmol/L okadaic acid), in tube 3 we added 20 µL of buffer 3 (buffer 2 containing 10 µmol/L CsA), and in tube 4 we added 20 µL of buffer 4 (buffer 2 containing 100 µg/L TRL). Buffer 4 has not been described before and was added to better evaluate samples from patients treated with TRL. We found no significant differences between buffers 3 and 4. The results were expressed in relationship to classic buffer 3. In addition, we measured the total activity of the substrate by counting 20 µL in a Beckman scintillation counter on the day of the assay, by measuring the spontaneous release via incubation of the substrate without lysate, and the capacity of AP (20 U) to dephosphorylate the substrate (maximum dephosphorylation).
The mixture was incubated for 30 min at 30 °C, and the reaction was stopped by the addition of 0.8 mL of 100 mmol/L K2HPO4 containing 50 g/L trichloroacetic acid. We then added 200 µL of cation-exchange resin (AG50W-X8, 100–200 mesh) and shook the suspension for 30 min at room temperature.
Cation-exchange resin was prepared in a batch procedure as follows. We suspended 2 g of dry resin was in 50 mL of water. After settling, the water was decanted and replaced with 5 mL of 1 mol/L NaOH. After mixing and settling, the supernatant was decanted completely and replaced with 10 mL of 1 mol/L HCl. After mixing and settling, the supernatant was again decanted, and the resin was washed with 20 mL of water. After removal of this supernatant, the resin was suspended in 2.2 mL of water and stored at 4 °C.
After the incubation, we centrifuged for 2 min at 12 000g. The released inorganic phosphate contained in the supernatant (500 µL) was measured by scintillation counting (18).
The initial results indicated the need to introduce an internal value to minimize the variability among different batches of 32P-labeled peptide and the radioactive decay. The internal value was defined as the capacity of AP to dephosphorylate the synthetic peptide, and it was considered to be the maximum dephosphorylation that could be observed with the peptide batch in use. The internal value was introduced in all remaining experiments. The results for CNA were expressed as: percentage of AP = 100 (cpm released by the sample)/cpm released by the AP). CsA-resistant phosphatase values (standard method) or TRL-resistant phosphatase values (buffer 4) were subtracted from the phosphatase activity in presence of okadaic acid and CNA (PP2B) was expressed as percentage of AP.
The interassay and intraassay CVs for measurement of CNA were 8% and 5%, respectively. CNA was assessed by individuals who were blinded to the treatment allocation of the patient.
measurement of il-2 and ifn- production
IL-2 and IFN- production was measured in treated patients and in the NHC group in the morning predose and 2 h postdose. IL-2 and IFN- production was assessed by individuals who were blinded to the treatment allocation of the patient.
We measured the IL-2 and IFN- production in 950 µL of whole blood incubated with 50 µL of PHA (1 g/L) and shaken for 5 h at 37 °C. At the end of the incubation, the samples were centrifuged in an Eppendorf microcentrifuge for 2 min, and the supernatant was removed and stored at -80 °C until assayed. IL-2 and IFN- concentrations were measured by ELISA. The detection limits were 3 ng/L for IL-2 and 80 IU/L for IFN- (10).
measurement of blood concentrations of CsA and trl
CsA concentrations in whole blood were determined by Emit,® with a specific monoclonal antibody, on a Cobas Mira automated analyzer (Dade-Behring) as described previously (19). TRL monitoring was carried out by MEIA assay with an IMx analyzer (Abbott) (20). Blood samples for determining the CsA and TRL blood concentration–time curve AUCs were drawn predose (cmin) and at 1, 2, 4, 6, 8, 10, and 12 h after the morning dose from stable, treated renal patients. AUCs were calculated by the linear trapezoidal rule.
measurement of plasma concentrations of mycophenolic acid
Plasma concentrations of mycophenolic acid (MPA) were analyzed by a validated HPLC-ultraviolet detection method described elsewhere (21)(22). The total run time was 12 min. The working range for MPA was 0.10–50 mg/L, and the within- and between-run CVs ranged from 4.5% to 9.7%.
International CsA, TRL, and MPA Testing Scheme Samples were also analyzed as external controls (from D.W. Holt, European Quality Control, London, UK).
statistics
Unless specified, all results are expressed as the median and the 25th–75th percentiles or the confidence interval. In some graphs, the 25th and 75th percentiles are shown. Statistical differences between groups were assessed using the nonparametric Mann–Whitney test. Differences are indicated on the graphs with an asterisk. Correlations between variables were assessed by another nonparametric test, the Spearman test (
), with SPSS statistical software (SPSS Inc.). P <0.05 was considered significant.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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cna measurements in vitro
Measurement of CNA was validated in vitro. PBMCs from healthy donors were cultured in the presence of increasing concentrations of CsA (0–400 µg/L) or TRL (0–50 µg/L). After 24 h, cells were lysed, and the CNA was evaluated. The results obtained showed inhibitions >75% when the CsA concentration in cultures was >100 µg/L and the concentration of TRL was >12.50 µg/L (Fig. 1
). Inhibitory concentrations may differ in vivo because of a clear difference between the free fraction of the immunosuppressant in biological fluids and in culture medium.
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cna measurements in patients
CNA was evaluated at 0 and 2 h postdose in the PBMCs of stable renal transplanted patients treated with CsA, TRL, MMF, CsA+MMF, and TRL+MMF (Fig. 2
).
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The median (25th–75th percentiles) CNA was 37.45 (21.32–56.15)% AP in the NHC group and 24.20 (17.72–35.58)% AP in the MMF group. These values were significantly lower in the CsA group than in NHC or MMF group, both predose [0 h; 6.42 (5.47–10.01)% AP] and 2 h postdose [3.87 (3.00–6.85)% AP; P <0.01; Fig. 2A
]. We observed similar values in the CsA+MMF group at 0 h [6.41 (4.88–12.22)% AP] and 2 h [3.90 (1.78–5.19)% AP; Fig. 2A
]. The CNA was significantly lower in the TRL group than in the NHC and MMF groups [0 h, 8.66 (6.14–23.94)% AP; 2 h, 5.68 (3.02–16.00)% AP; P <0.01; Fig. 2B
] and the TRL+MMF group [0 h, 18.53 (7.63–26.62)% AP; 2 h, 11.80 (4.06–14.63)% AP; P <0.01], although TRL patients displayed more variability than CsA patients. We found no significant differences in CNA between patients treated with CNIs alone and those treated with CNIs+MMF (Fig. 2
).
il-2 and ifn- production in whole blood from patients
IL-2 and IFN- production after in vitro stimulation of whole blood with PHA was evaluated in the study groups (Figs. 3
and 4
). Results showed considerable variability in IL-2 production in the NHC and MMF groups. IL-2 production was lower 2 h postdose in all groups other than the NHC group (Fig. 3
). The production of IL-2 postdose in the CsA or TRL+MMF groups [median (25th–75th percentiles), 166.48 (81.06–377.01) ng/L and 122.13 (51.02–180.00) ng/L, respectively] was lower than in the CsA- or TRL-alone groups [median (25th–75th percentiles), 276.52 (190.41–385.25) ng/L (P <0.001) and 249.34 (127.48–363.50) ng/L (P <0.001) respectively; Fig. 3
].
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We observed no predose differences in IFN- production between the control group and patients treated only with CNIs [median (25th–75th percentiles), 22.39 (9.78–34.34) IU/mL vs 29.25 (25.55–31.90) IU/mL]. Postdose, blood from the CsA, TRL, and TRL+MMF groups showed clear inhibition compared with blood from the NHC group (Fig. 4
).
pd and pk correlations
The correlation between the classic PK parameters and PD indices was studied with the nonparametric Spearman test (). P <0.05 was considered significant (Table 2
).
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There was a significant inverse correlation (r >0.7) between CNA 2 h postdose and the AUC for all groups studied and for CNA 2 h postdose and c2 h, especially in the CsA and CsA+MMF groups.
IL-2 production 2 h postdose and c2 h also were significantly inversely correlated in the CsA, CsA+MMF, and TRL+MMF groups, as were IL-2 production 2 h postdose and AUC in the TRL+MMF group.
There was no correlation between IFN- and cmin in any group. In contrast, IFN- production was significantly inversely correlated with c2 h and AUC in the CsA group (Table 2
).
To identify the redundancy among PD indicators, we studied the correlations between the pharmacodynamics at different times and the various PD indicators (Table 3
). We found a strong correlation between CNA 0 h predose and CNA 2 h postdose in all groups (r = 0.93–0.96). We also found a significant correlation between IL-2 production 0 h predose and IL-2 production 2 h postdose in the CsA groups (r = 0.92–0.96) and the TRL groups (r = 0.75–0.82; Table 3
).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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This study included PD markers such as CNA in PBMCs and IL-2 and IFN- production in whole blood along with classic PK parameters, and compared them in stable renal transplant patients treated with CsA or TRL alone or in combination with MMF.
We decided to measure CNA in PBMCs and not in whole blood, as other authors have done (23), for several reasons: Our main interest was the impact of the drug on enzyme activity inside the lymphocytes, which are the main producers of relevant interleukins involved in the alloresponse. This impact probably depends on the equilibrium between uptake, cyclophilin binding, P-glycoprotein-dependent excretion, and probably other factors related to each cell type. In our opinion the use of whole blood to measure CNA has two drawbacks: (a) the CN of lymphocytes represents only a small part (<10%) of the whole-blood CN (23), and thus the greater part of the CNA measured when whole blood is used is not relevant; and (b) more importantly, the technique involves a cell disruption step in which the CsA or TRL stored in red blood cells becomes available for lymphocyte CN. In vivo, only intralymphocytic CNIs are available for lymphocyte CN; therefore, the measurement of CNA in whole blood does not reflect the real impact of inhibitors on the immune system.
However, to evaluate IL-2 and IFN- production, we decided to use whole blood because with the technique described by Stein et al. (10) the lymphocyte microenvironment was maintained throughout the assay time (5 h at 37 °C), and intracellular concentrations changes in CNIs were avoided.
The profile chosen to evaluate PD indices was predose and 2 h postdose for all groups. Although the CsA peak is reached 1 h after drug administration, it is generally agreed that CsA concentrations at 2 h (c2 h) correlate better with AUC, cmax, and clinical impact. In fact, c2 h values are currently used to adjust CsA concentrations in clinical practice (24)(25). In addition, Caruso et al. (23) showed that the nadir of CNA in PBMCs was at 3 h postdose and that CNA values were very similar at 1, 2, 3, and 5 h postdose. Those authors noted that there was a delay between cmax and the nadir of CNA in PBMCs.
Our results confirm previous reports (4)(5) that measurement of CNA in the PBMCs of patients treated with CsA is feasible with the technique described by Fruman et al. (18). Our findings are similar to those reported by Batiuk et al. (5), Kung et al.(26), and Halloran et al. (27), who did not observe complete inhibition of CNA in vivo in CsA- and TRL-treated groups.
The inhibition of CNA observed in the CsA and TRL groups compared with the NHC group (83% and 77%, respectively, at 0 h; 90% and 85% at 2 h postdose) was higher than in previous reports. This is probably attributable to our decision to express the CNA results as percentages of the capacity of an AP to release 32P from the synthetic peptide, which in our opinion increases the stability of this marker by minimizing the variability attributable to differences in the batches of 32P-labeled peptides and radioactivity decay between assays.
The variability of CNA in TRL-treated patients was higher than in those receiving CsA. Some authors attribute this variability to the fact that the CsA–cyclophilin complex has better accessibility to the active site of the synthetic peptide used (27). Others account for these differences by pointing to the existence of a limiting factor, the active immunophilins present in the lymphocytes; it appears that active FKBPs have a greater limiting effect than active cyclophilins (26). In addition, by working with disrupted cells we introduced conditions that are different from those in intact cells, above all in the distribution of certain immunophilins in the various compartments in the lymphocyte. In other words, we may have altered the probability of the formation of the immunosuppressant–immunophilin active complex (CsA–cyclophilin and TRL–FKBP). However, when we used a buffer with TRL in the phosphatase assay (buffer 4; described in Materials and Methods) there was no significant change in the results.
In combined therapies with CNIs and MMF, the CNA measurement does not directly reflect the additional immunosuppressive effect of MMF. The measurement of IL-2 production, in contrast, seems to reflect the effect provided by the presence of MMF in CNI therapies, especially postdose. In the NHC group, IL-2 production was highly variable. Variability in the CNI-treated groups was lower, especially postdose. Because of the short in vitro culture time used, IL-2 production depended on the number of preactivated cells present in vivo in each individual. The decrease in variability in the CNI-treated patients postdose indicates that the variability in the number of preactivated cells present in the patients does not disqualify IL-2 production as an indicator of the CNI effect. However, it is likely that longer in vitro incubation times could reduce this variability.
The CNA in patients receiving monotherapy with MMF was not statistically different from that in the NHC control, but it was higher than in patients treated with CNIs.
The decrease in IL-2 production in patients receiving MMF in addition to CNIs in comparison with those receiving CNI alone was probably attributable to the inhibitory effect of MMF on clonal expansion of activated lymphocytes. The decrease in the number of active lymphocytes, rather than a direct effect of MMF on CNA or in the production of IL-2 and IFN-, was probably responsible for the decrease in production.
IFN- production is highly variable in this population. There was a lack of correlation between cmin and IFN- at 0 h, indicating that IFN- is not a useful marker for these studies.
IL-2 production was lower in the CsA+MMF group than in the CsA-alone group at 2 h. These differences can not be explained by differences in the CsA concentrations: there was no significant differences in CsA dose [1.93 (0.50) vs 2.60 (0.90) mg · kg-1 · day-1, respectively]. We found only small difference in the cmin values [median (25th–75th percentiles), 61.50 (39.50–92.50) vs 93.00 (80.75–128.25) µg/L, respectively], and some differences in the cmax were not statistically significant. No differences were evident in the CsA concentrations at 2 h postdose [median (25th–75th percentiles), 494.50 (251.00–654.25) and 481.00 (318.75–561.75) µg/L, respectively]; therefore, the presence of MMF seems to be responsible for the additional inhibitory effect on IL-2 production seen at 2 h in the CsA+MMF group.
IL-2 production was lower in the TRL+MMF group than in the TRL-alone group at 2 h. Those differences cannot be explained by differences in the TRL concentrations: neither the doses nor cmin showed significant differences. The c2 h was even lower in the TRL+MMF group than in the TRL-alone group [median (25th–75th percentiles), 13.50 (11.70–17.10) and 21.90 (10.35–33.15) µg/L, respectively]; therefore, the presence of MMF seems to be responsible for the additional inhibitory effect on IL-2 production seen at 2 h in the TRL+MMF group as well.
These two results suggest that the addition of MMF to CNI therapies increases the inhibition of IL-2 production, although it does not directly affect CNA. The reason could be that reducing the clonal expansion of activated T cells reduces the number of cells ready to produce IL-2 in our in vitro assay.
Several strategies have been proposed in the past to monitor the pharmacodynamics of CNI immunosuppressants. Here we compared some of them in monotherapy and combined therapies to identify the most useful ones to monitor the biological impact of new combinations of immunosuppressants, different doses from those considered standard, or the study of problematic patients.
In our study, the PD indicator with the best correlation with AUC and c2 h was CNA, followed by IL-2 production. In general, IFN- had the poorest correlation with PK parameters; the correlation was particularly poor between IFN-0 h and cmin. IL-2 production, however, seems to reflect the additional immunosuppressive effect introduced by MMF in combined therapies. This additional effect was not detected by CNA determination.
There was a very good correlation with CNA at 0 and 2 h for both CsA and TRL and with IL-2 at 0 and 2 h, especially for CsA.
In conclusion, the measurement of CNA may be a good predictor of the immunosuppression caused by CsA or TRL monotherapies, whereas IL-2 production is potentially more useful for monitoring combined therapies comprising CNIs and MMF.
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Acknowledgments |
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Footnotes |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  C. Sommerer, T. Giese, S. Meuer, and M. Zeier Pharmacodynamic monitoring of calcineurin inhibitor therapy: Is there a clinical benefit? Nephrol. Dial. Transplant., January 1, 2009; 24(1): 21 - 27. [Full Text] [PDF]
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, 0 h predose; 
, 2 h postdose. *, P <0.05 compared with the NHC group. The results are expressed as % AP.
, predose;