- Email: [email protected]
Monitoring Biological Action of Rapamycin in Renal Transplantation
Transplantation Monitoring Biological Action of Rapamycin in Renal Transplantation Domenica Leogrande, MD,1 Annalisa Teutonico, MD,1 Elena Ranieri, PhD,2 Marilisa Saldarelli, PhD,1 Loreto Gesualdo, MD,3 F. Paolo Schena, MD,1 and Salvatore Di Paolo, MD1 Background: Inhibition of P70S6 kinase (P70S6K) phosphorylation in activated T cells is 1 of the major mechanisms by which rapamycin exerts its immunosuppressive action. Study Design: Observational cohort study. Settings & Participants: 2 different groups of kidney transplant recipients at a single center: 30 transplant recipients converted from mycophenolic acid and low-dose prednisone plus cyclosporine A to mycophenolic acid and low-dose prednisone plus rapamycin therapy for chronic allograft nephropathy (group 1) and 16 recipients of suboptimal organs converted from tacrolimus plus rapamycin to rapamycin therapy alone after 3 months (group 2). Predictor: Exposure to rapamycin therapy and rapamycin trough levels. Outcomes & Measurements: Basal and stimulated phosphorylation of P70S6K was measured by using Western blotting in patients’ peripheral-blood mononuclear cells before and 6 to 11 months after conversion to rapamycin-based therapy. Kinase activation was attained in vivo by means of intravenous insulin injection. Results: The potency of rapamycin inhibition of P70S6K phosphorylation varied among patients (RAPA blood concentration required to achieve 50% inhibition of P70S6K activation for mitogenactivated kinase, 3.14 to 12.14 ng/mL) and failed to correlate with drug trough levels. The combination of tacrolimus and rapamycin limited the inhibitory effect of the latter drug on P70S6K activation. Limitations: Need for additional studies exploring the relationship between P70S6K activity and kidney graft outcome. Exclusion of patients with diabetes. Conclusions: Long-term rapamycin treatment inhibits P70S6K phosphorylation in peripheral-blood mononuclear cells without significant correlation with rapamycin trough levels. By measuring in vivo the biological action of rapamycin, the assay may provide potentially relevant information for the clinical management of rapamycin-treated patients. Am J Kidney Dis 50:314-325. © 2007 by the National Kidney Foundation, Inc. INDEX WORDS: Kidney transplantation; rapamycin; tacrolimus; P70S6 kinase; peripheral-blood mononuclear cells; in vivo assay.
R
apamycin (RAPA) is a novel immunosuppressive drug recently approved for use in renal transplant recipients. RAPA penetrates the plasma membrane and binds to FK-binding protein 12 (FKBP-12) to form a complex that binds
From the 1Department of Emergency and Organ Transplants, Division of Nephrology, Dialysis and Transplantation, University of Bari, Policlinico, Bari; 2Department of Biomedical Sciences, Section of Clinical Pathology; and 3 Department of Biomedical Sciences, Division of Nephrology, University of Foggia, Foggia, Italy. Received January 26, 2007. Accepted in revised form May 14, 2007. Originally published online as doi: 10.1053/j.ajkd.2007.05.002 on July 9, 2007. *Present address: Division of Nephrology and Dialysis, Hospital “Dimiccoli,” Barletta, Italy Address correspondence to Salvatore Di Paolo, MD, Division of Nephrology, Hospital “Dimiccoli,” ASL BAT, Viale Ippocrate, 1-Barletta, Italy. E-mail: [email protected] © 2007 by the National Kidney Foundation, Inc. 0272-6386/07/5002-0017$32.00/0 doi:10.1053/j.ajkd.2007.05.002 314
to the mammalian target of RAPA (mTOR).1 This interaction causes dephosphorylation and inactivation of P70 ribosomal S6 kinase (P70S6K), which, when activated, stimulates protein synthesis and cell-cycle progression (Fig 1).1 Both animal and clinical studies showed a relationship between RAPA trough concentrations and graft outcome.1-3 Nonetheless, dose adjustments for such critical-dose drugs as RAPA that rely only on measuring blood concentrations may not necessarily correlate with pharmacological effects of the drugs on immune cells. Thus, identification of molecular markers enabling the assessment and follow-up of biological effects of RAPA may help optimize drug therapy and evaluate new immunosuppressive multidrug regimens. Previous studies investigated the immunosuppressive properties of RAPA by using in vitro or ex vivo experimental models.4-7 Thereafter, a few investigations suggested that the pharmacodynamic effects of RAPA derivatives could be
American Journal of Kidney Diseases, Vol 50, No 2 (August), 2007: pp 314-325
P70S6 Kinase Inhibition and Rapamycin Efficacy
315
Figure 1. A simplified model of the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K)-AKT pathways implicated in the control of T-cell proliferation. Engagement of the T-cell receptor (TCR) and such costimulatory receptors as CD28 elicits expression and secretion of the T-cell growth factor interleukin 2 (IL-2) and expression of its high-affinity receptor (IL-2R), rendering the cells competent for IL-2– driven proliferation. The TCR and CD28 also can control cell-cycle progression independently of IL-2 by direct activation of the PI3K/AKT/mTOR pathway. Similarly, various peptide growth factors, such as insulin, bind to specific cell-surface receptors (I-R) and thereby activate the PI3K/AKT/mTOR pathway. The GL-raptor-mTOR complex controls messenger RNA (mRNA) translation through phosphorylation of P70S6K and eIF4E-binding protein 1 (4EBP1). Activation of P70S6K leads in turn to phosphorylation of the 40S ribosomal protein S6, whereas phosphorylation of 4EBP1 releases the eukaryotic initiation factor 4E (eIF4E) to restore cap-dependent translation. In addition, raptor-mTOR facilitation of G1 to S cell-cycle transition is mediated, at least in part, by the increased translation of mRNAs encoding positive regulators of cell-cycle progression, such as cyclin D3, cyclin E, and c-Myc, and by decreased translation of negative regulators thereof, such as the cyclin-dependent kinase inhibitor p27kip1. Rapamycin (RAPA) binds to FK-binding protein 12 (FKBP-12) and leads to cell-cycle arrest in G1 phase through the inhibition of all these effects of raptor-mTOR, but mainly blocking the phosphorylation of P70S6K and 4EBP1.
316
Leogrande et al
determined reliably in vivo by monitoring P70S6K activity in peripheral-blood mononuclear cells (PBMCs) of patients with cancer.8,9 Next, Hartmann et al10 described pronounced inhibition of basal P70S6K phosphorylation in PBMCs isolated from RAPA-treated kidney transplant recipients and suggested that the phosphorylation status of the kinase can provide more relevant information than RAPA trough levels to prevent acute allograft rejection. Finally, we recently showed that monitoring P70S6K phosphorylation can help predict and monitor regression of cancer lesions in renal transplant recipients with Kaposi sarcoma who were converted to RAPA therapy and possibly adjust the biologically active doses of the mTOR inhibitor.11 The present study is designed with the following aims: (1) to determine whether long-term administration of RAPA in vivo is associated with inhibition of basal and stimulated P70S6K phosphorylation, as shown in vitro; and (2) to assess whether RAPA blood trough concentration, the commonly used index to monitor drug dosing, correlates with degree of P70S6K phosphorylation (ie, activation)12 in kidney transplant recipients on maintenance immunosuppression therapy with RAPA. As a secondary aim, we also investigated possible drug interactions between RAPA and the calcineurin inhibitor tacrolimus in patients treated using a combination of the 2 immunosuppressive drugs. METHODS Patients Two different groups of kidney transplant recipients were investigated.
Group I Starting January 2002 to December 2004, all patients who received a kidney transplant, were 12 to 60 months after transplantation, had stable graft function and serum creatinine levels less than 2.5 mg/dL (⬍221 mol/L), and were in treatment with cyclosporine A microemulsion (CsA), mycophenolate mofetil (1 to 2 g/d), and low-dose steroids (prednisone, 2.5 to 5 mg/d) were offered a protocol graft biopsy. Patients for whom the histological diagnosis of chronic allograft nephropathy13 was made were requested to be converted to RAPA therapy without further modification of the remaining immunosuppressive therapy. Exclusion criteria were age younger than 18 or older than 60 years, histological evidence of recurrent or de novo renal disease, diagnosis of diabetes at any time in their clinical history, significant coexisting severe disease (cardiac, hepatic, or
neoplastic), absolute need for drugs interfering with glucose metabolism,14,15 fasting cholesterol level greater than 300 mg/dL (⬍7.76 mmol/L), and/or triglyceride level greater than 350 mg/dL (⬎3.95 mmol/L). We assessed 232 potential subjects; 102 had already been recruited in different clinical trials, and 22 refused a protocol biopsy. At histological examination, the diagnosis of chronic allograft nephropathy was made in 74 of the remaining 108 patients. Of these, 5 subjects were excluded for the presence of recurrent or de novo glomerular disease, and 19 patients, for diabetes (7 patients), severe hepatic (6 patients) or cardiac (2 patients) disease, or marked hyperlipemia (4 patients). Three transplant recipients underwent investigation for comorbidity, and 8 patients refused to change their immunosuppressive regimen. We eventually recruited 39 patients who started RAPA therapy 12 to 16 hours after the abrupt discontinuation of CsA therapy. All transplant recipients were administered a single oral loading dose of RAPA (0.1 mg/kg), followed by a daily dose of 5 mg. Whole-blood RAPA trough concentration was measured first on day 5 after the conversion, and RAPA daily dose was modified to achieve target trough levels of 8 to 12 ng/mL. All patients were studied immediately before the discontinuation of CsA therapy and 9 to 11 months after the conversion to RAPA therapy.
Group II In the same period at our center, some recipients of suboptimal kidneys16 were randomly assigned to a calcineurin inhibitor–sparing regimen comprised of tacrolimus (target trough level, 6 to 8 ng/mL), RAPA (target trough level, 4 to 8 ng/mL), and low-dose steroids for the first 3 months after grafting, then discontinued tacrolimus therapy and increased RAPA target trough levels to 8 to 12 ng/mL. Collectively, 28 of 93 transplant recipients of suboptimal kidneys were assigned to this regimen; the others entered different clinical trials.16 One patient experienced an acute rejection episode in the first 3 months after engraftment and was converted to full-dose tacrolimus therapy, 2 patients developed posttransplantation diabetes, 1 patient had severe hepatitis C virus–related hepatic disease, 3 patients had marked hypertriglyceridemia, and 1 patient showed progressive graft function deterioration. Therefore, we recruited 20 patients who were examined immediately before and 6 to 8 months after conversion to RAPA-alone therapy. Finally, 7 patients with stable graft function on maintenance therapy with CsA, mycophenolate mofetil, and lowdose steroids were studied twice, with an interval of approximately 6 months, without modification of immunosuppressive drugs, and served as controls to evaluate the spontaneous modification over time of the parameter tested. All patients were asked to give their written informed consent to participate in the study, according to the Guidelines of the Local Ethical Committee.
In Vitro Activation of P70S6K mTOR signaling appears to be regulated downstream of phosphatidylinositol-3-kinase/AKT on T-cell receptor engagement or cell stimulation with lymphokines, growth factors, and hormones (eg, insulin; Fig 1).1,15,17 Preliminary
P70S6 Kinase Inhibition and Rapamycin Efficacy
317
dose–response experiments showed that maximal activation of P70S6K in control PBMCs was achieved 15 minutes after stimulation with 10 nmol/L of insulin, ie, the same order of magnitude of peak serum insulin after intravenous administration. To explore the inhibitory effect of RAPA on P70S6K activation, PBMCs were preincubated for 30 minutes with serial dilutions of RAPA (0 to 10 ng/mL) before the addition of 10 nmol/L of insulin.
quantification, defined as the lowest concentration of drug that could be assayed with a good level of precision and inaccuracy, at 2.5 ng/mL.
In Vivo Activation of mTOR Pathway
Percent inhibition ⫽ [1 ⫺ (Treatment/Pretreatment)] ⫻ 100
To evaluate in vivo the activation of P70S6K and its inhibition by RAPA, 0.1 IU/kg of body weight of soluble insulin was injected intravenously into patients in the fasting state 12 or 24 hours after the last dose of the calcineurin inhibitor or RAPA, respectively.14,15 Blood samples (25 mL) were drawn immediately before and 15 minutes after insulin administration.
Western Blotting Human PBMCs were isolated, lysed, and subjected to blotting as described.11,15 Briefly, 50 g of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene difluoride membrane. The filter was probed with mouse monoclonal antiphospho-P70S6K antibody, raised against a peptide that contained the phosphorylated Ser-411 (Saint Cruz Biotechnologies, Santa Cruz, CA) at 1:100 dilution. Membranes then were washed and incubated with horseradish peroxidase–conjugated goat antimouse immunoglobulin G. To measure total (nonphosphorylated and phosphorylated) kinase, the same membranes were then stripped and immunoblotted with antihuman P70S6K monoclonal antibody raised against a peptide mapping at the carboxy terminus of P70S6K (Saint Cruz Biotechnologies). Cell lysates from each patient were processed on the same gel. The enhanced chemiluminescence system (Amersham Biosciences, Little Chalfont, UK) was used for detection according to the manufacturer’s instructions. The intensity of signals detected by means of enhanced chemiluminescence was quantitated by using densitometric analysis, and results of phospho-P70S6K were expressed in arbitrary units (AU) after normalization to total P70S6K.
Pharmacokinetic Analysis Blood concentration of RAPA was determined by using a high-performance liquid chromatography assay with UV detection, as previously described.18 Application of the method in our laboratory was validated by a reference laboratory that had established an international proficiency testing control system for RAPA.19 The internal standard (32-desmethoxy RAPA) was obtained from Supelco, SigmaAldrich, Milan, Italy. Overall recovery was checked at 5 and 20 ng/mL. The recovery from blood precipitation with zinc sulfate tested was 75.3% ⫾ 3.2% for RAPA and 72.1% ⫾ 2.8% for internal standard, that from reversed-phase extraction was 86.5% ⫾ 3.0% for RAPA and 86.1% ⫾ 2.6% for internal standard. At a 2.5-ng/mL RAPA concentration, within-day and between-day coefficients of variation of the assay were 7.6% and 6.5%, respectively. Inaccuracy of the method was less than 9%. Thus, we set the lower limit of
Pharmacodynamic Analysis In patients in group I, the inhibitory effect of RAPA on P70S6K phosphorylation was calculated using the following formula:
where pretreatment represents the percentage of increase in P70S6K phosphorylation over basal after insulin injection in patients before RAPA therapy, and treatment represents the percentage of increase in P70S6K phosphorylation after longterm treatment with RAPA. The percentage of inhibition of basal P70S6K phosphorylation after RAPA therapy also was calculated. In patients in group II, we were instead interested in exploring the possible interference of tacrolimus on RAPAinduced inhibition of P70S6K activation. To this aim, we compared the percentage of increase in insulin-stimulated P70S6K phosphorylation normalized to RAPA trough level or RAPA daily dose in the presence and absence of the calcineurin inhibitor.
Statistical Analysis Differences between quantitative variables were tested by means of Mann-Whitney U test or Wilcoxon signed-rank test, as appropriate. The relationship between nonparametric variables was tested by using Spearman rank correlation. P less than 0.05 is considered statistically significant. The Statview software package (version 5.0; SAS Institute Inc, Cary, NC) was used for all analyses.
RESULTS
Patients
Thirty of 39 patients in group I completed the study (1 patient developed posttransplantation lymphoproliferative disease, 1 patient progressed to end-stage renal disease, 2 patients developed posttransplantation diabetes, 3 patients had serious unamenable side effects [arthralgias, abdominal pain, and diarrhea] that led to discontinuation of RAPA therapy, 1 patient declined his consent, and 1 patient was lost to follow-up). In group II, 2 patients developed posttransplantation diabetes and 2 patients had serious arthralgias after the conversion to RAPA-alone therapy and were switched to tacrolimus therapy. Consequently, we studied 16 patients. Throughout follow-up, care was taken to avoid any major modification of pharmacological therapy.14,15 In group I, 3 patients had a temporary decrease in mycophenolate mofetil dose at some time during the study because of transient
318
Leogrande et al Table 1. Anthropometric and Laboratory Features of Patients Examined at Study Start and End
Age (y) Sex (M/F) Time from transplantation (mo) Serum creatinine (mg/dL) Daily proteinuria (g/d) Creatinine clearance (mL/min) Triglycerides (mg/dL) Total cholesterol (mg/dL) Leukocytes (/L) Platelets (103/L) CsA/TAC blood levels (ng/mL) CsA/TAC daily dose (mg/kg) RAPA blood levels (ng/mL) RAPA daily dose (mg/kg) MMF dose (mg/d)
Group I
Group II
43.9 ⫾ 12.5 16/14 28.9 ⫾ 13.8
52.0 ⫾ 5.4 12/4 3
Start
End
Start
End
1.46 ⫾ 0.34 0.78 ⫾ 0.24 65.1 ⫾ 21.6 136.7 ⫾ 61.7 204.1 ⫾ 52.5 7,179 ⫾ 2,121 234 ⫾ 56 620.7 ⫾ 147.3 2.38 ⫾ 0.78 — — 1,183 ⫾ 334
1.59 ⫾ 0.42 1.08 ⫾ 0.46 62.9 ⫾ 19.7 201.3 ⫾ 79.5* 228.7 ⫾ 48.8† 6,712 ⫾ 1,814 219 ⫾ 49 — — 9.01 ⫾ 2.21 0.045 ⫾ 0.021 1,150 ⫾ 267
2.17 ⫾ 0.78 1.06 ⫾ 0.63 37.8 ⫾ 11.4 207.1 ⫾ 61.8 212 ⫾ 33.1 6,638 ⫾ 1,531 264 ⫾ 76 7.20 ⫾ 2.65 0.047 ⫾ 0.018 5.66 ⫾ 2.19 0.069 ⫾ 0.035 —
2.10 ⫾ 0.69 1.29 ⫾ 0.84 40.6 ⫾ 10.2 225. ⫾ 127.4 236.4 ⫾ 56.6 7,024 ⫾ 2,016 249 ⫾ 66 — — 8.82 ⫾ 1.62 0.053 ⫾ 0.024 —
Note: Cyclosporine A whole-blood levels were measured 2 hours after the morning dose, rapamycin and tacrolimus levels were obtained by monitoring predose (trough) levels. Results expressed as mean ⫾ SD. To convert serum creatinine in mg/dL to mol/L, multiply by 88.4; creatinine clearance in mL/min to mL/s, multiply by 0.01667; serum cholesterol in mg/dL to mmol/L, multiply by 0.02586; serum triglycerides in mg/dL to mmol/L, multiply by 0.01129. Abbreviations: CsA, cyclosporine A; TAC, tacrolimus; RAPA, rapamycin; MMF, mycophenolate mofetil. *P ⫽ 0.001. †P ⫽ 0.04.
leukopenia, whereas 2 patients permanently decreased their dose from 2 to 1.5 g of mycophenolate mofetil for abdominal pain and leukopenia. No patient studied experienced an acute rejection episode or clinically relevant infection or showed significant modifications in renal function. The main features of patients in each group are listed in Table 1. Most patients showed stable RAPA blood levels over time; the greatest variability was associated with the first 4 to 8 weeks after the start of RAPA therapy.
sponse to the inhibitory effect of the mTOR inhibitor, such that the RAPA concentration required to achieve 50% inhibition of P70S6K activation (IC50) ranged from 0.6 to 4.1 ng/mL among different donors (n ⫽ 4; Fig 2C). Finally, control PBMCs were cultured for 2 to 48 hours in the presence or absence of 200 ng/mL of CsA, then challenged with 10 nmol/L of insulin. The calcineurin inhibitor failed to affect basal or activated phosphorylation of P70S6K, as already reported for AKT activation.15
In Vitro Experiments
Group I In the absence of RAPA, intravenous insulin injection caused a 241.8% ⫾ 94.7% increase in P70S6K phosphorylation over basal levels (Fig 3A and B). Long-term exposure to RAPA was associated with a significant decrease in mean levels of both basal and insulin-stimulated P70S6K phosphorylation without modification of total P70S6K content (Fig 3A and B). Next, we calculated the percentage of change from baseline of phospho-P70S6K, basal and stimulated, in each patient to ensure more accurate assessment of drug-induced changes (Fig 3C).
First we measured the coefficient of variation (CV) of kinase phosphorylation in healthy controls. CVs, calculated as SD/mean of phosphoP70S6K, were 39% for basal and 68% for activated P70S6K phosphorylation (n ⫽ 5). RAPA (10 ng/mL) did not modify basal P70S6K phosphorylation in control PBMCs (not shown). Instead, the mTOR inhibitor significantly downregulated insulin-stimulated phosphorylation of P70S6K in a dose-dependent fashion (Fig 2B and C). However, we observed very large intersubject variability in cell re-
In Vivo Study
P70S6 Kinase Inhibition and Rapamycin Efficacy
319
Figure 2. In vitro activation of P70S6K by insulin and kinase inhibition by rapamycin (RAPA). (A) Time-course of P70S6K phosphorylation by 10 nmol/L of human regular insulin in human peripheral-blood mononuclear cells (PBMCs) isolated from healthy donors. (B, C) Dose-dependent inhibition of insulin-induced activation of P70S6K phosphorylation by RAPA. (B) Representative Western blotting from 1 healthy individual. Kinase activation by 10 ng/mL of interleukin 2 (IL-2) is also represented to the aim of comparison. (C) Quantitation of inhibition of kinase by RAPA (n ⫽ 4). *P ⬍ 0.05; **P ⬍ 0.0001 (versus insulin-treated cells).
RAPA IC50 for mitogen-activated phosphorylation showed large interpatient variability (median, 6.6 ng/mL; range, 3.14 to 12.14 ng/mL). Of note, the degree of inhibition of insulinstimulated P70S6K phosphorylation failed to correlate significantly with RAPA trough levels or daily dose of the mTOR inhibitor (Fig 4). The IC50 of basal P70S6K phosphorylation was strikingly high with a median of 9.35 ng/mL, with very large interpatient variability (3.7 to 21.1 ng/mL). Finally, we wondered whether inhibition of P70S6K phosphorylation would show dose dependence in individual patients. Thus, we reexamined 4 patients after approximately 6 months and found a stable relationship between RAPA trough level and percentage of inhibition of insulinstimulated P70S6K activation, with a CV of per-
centage of inhibition (expected versus measured) of 12.4% (Fig 5). The intra-assay CV for P70S6K phosphorylation was steadily less than 9%. Intraindividual variabilities, tested in 7 controls during a time span of 6 months, were 12.2% and 14.1% for basal and insulin-activated P70S6K phosphorylation, respectively. Instead, the kinase showed large interindividual variability; CVs of basal and stimulated phosphorylation were 47.8% and 38.1%, respectively (n ⫽ 30 patients). Group II Patients in this group were examined first 3 months after engraftment and thereafter were reevaluated 6 months after discontinuation of tacrolimus therapy (Table 1). We sought for a possible interference of tacrolimus on mTOR
320
Leogrande et al
Figure 3. In vivo effect of long-term rapamycin (RAPA) treatment on basal and insulin-stimulated phosphorylation of P70S6K in human peripheral-blood mononuclear cells (PBMCs). Thirty kidney transplant recipients were studied immediately before and 9 to 11 months after discontinuation of cyclosporine A and conversion to RAPA therapy. In vivo activation of the target kinase was obtained by intravenous administration of 0.1 U/kg of human regular insulin. (A) Representative immunoblots: for each patient, all cell lysates were processed on the same gel. (B) Quantification of pP70S6K before and after 9 to 11 months of treatment with RAPA. Results expressed as mean (⫾SD) of absolute values measured in each patient. (C) Change (⌬) in basal and insulin-stimulated P70S6K phosphorylation after long-term RAPA therapy. Data represent mean (⫾SD) of percentage of change from baseline measured in each patient. *P ⬍ 0.0001 versus pretreatment values.
inhibition by RAPA. To this aim, we compared the percentage of increase in insulin-stimulated P70S6K phosphorylation, normalized to either RAPA trough level or RAPA daily dose, in the presence and absence of tacrolimus. As shown in Fig 6, withdrawal of tacrolimus was associated with greater inhibition of kinase activation by RAPA in each individual patient. This suggests that the combination of tacrolimus and RAPA limits the inhibitory effect of the latter drug on mitogen-induced P70S6K phosphorylation.
We also confirmed the lack of relationship between degree of P70S6K phosphorylation in PBMCs and RAPA trough level in this group of patients (Table 2). DISCUSSION
This study shows the ability of RAPA to inhibit in vivo P70S6K activation in PBMCs of renal transplant recipients. Unexpectedly, such inhibition failed to correlate with whole-blood RAPA trough levels. Next, we showed that ta-
P70S6 Kinase Inhibition and Rapamycin Efficacy
321
Figure 4. Lack of a relationship between inhibition of insulin-stimulated phosphorylation of P70S6K and either rapamycin trough level or drug daily dose in patients converted from therapy with cyclosporine to the mTOR inhibitor (group I).
crolimus apparently dampened the inhibitory effect of RAPA on P70S6K phosphorylation. Monitoring RAPA blood levels is recommended to optimize therapy on account of the narrow therapeutic ranges of RAPA, wide intraindividual and interindividual variations in its pharmacokinetics, and potential for significant drug-drug interactions. However, pharmacokinetics cannot account for intersubject variability in the sensitivity to immune suppression by similar blood concentrations of RAPA and cannot measure the biological effect of the drug on immune cells. The selective blockade of the P70S6K acti-
vation cascade by RAPA was shown to efficiently inhibit interleukin 2– and mitogeninduced S phase entry and subsequent T-cell proliferation (Fig 1), resulting in immunosuppression,20 which supports the use of phosphorylation status of P70S6K as a biomarker for mTOR inhibition in RAPA-treated patients. Previously, only a few studies explored the inhibitory effect of RAPA on human lymphocyte function by using in vitro assays in which whole human blood or isolated lymphocytes were exposed to the test drug and mitogens.4,5,7 These investigations reported RAPA IC50 values of at
322
Leogrande et al
Figure 5. Longitudinal assessment of P70S6K activation in 4 individual patients. Results expressed as both (upper panel) absolute values of insulin-stimulated P70S6K phosphorylation and (lower panel) change (⌬) versus pretreatment levels in each patient. Figures over each column represent rapamycin trough levels (nanograms per milliliter) at the time of the assay.
least 30 nmol/L (27.3 ng/mL),5,7 which are definitely greater than the IC50 values reported here and largely greater than the therapeutic range of RAPA. Next, Yatskoff and Gallant4 described an in vitro P70S6K assay on isolated human lymphocytes in which less than 25% inhibition was achieved at a RAPA concentration of 50 ng/mL. In our in vitro model, P70S6K activation was inhibited by RAPA in a dose-dependent fashion, although with large intersubject variability, with an IC50 range of 0.6 to 4.1 ng/mL. A major problem with the use of in vitro studies is that the dose and/or concentration required to inhibit a biological target may not necessarily be similar to the concentration required in vivo. The striking discrepancies among results reported previously apparently corrobo-
rate the difficulty extrapolating findings from in vitro or ex vivo studies to the clinical setting. Hartmann et al10 recently explored the ability of RAPA to inhibit P70S6K basal activation in PBMCs from renal transplant recipients in vivo and suggested that RAPA trough levels of 6 ng/mL or greater adequately suppressed kinase phosphorylation; a cutoff value of 60% phosphorylation relative to controls was highly predictive of acute rejection. However, the study extrapolated the percentage of basal P70S6K phosphorylation inhibition by comparison to the phosphorylation signal of healthy controls. Unfortunately, we and others also found large intersubject variability in basal P70S6K phosphorylation in healthy donors,8,9 similar to immunosuppressed patients,10 and this would greatly limit the reliabil-
P70S6 Kinase Inhibition and Rapamycin Efficacy
323
Figure 6. Insulin-stimulated P70S6K phosphorylation in 16 renal transplant recipients while treated with rapamycin (RAPA) plus low-dose tacrolimus (⫹) and 6 months after conversion to RAPAalone therapy (⫺). Percentage of increase in P70S6K phosphorylation over basal is normalized to either RAPA trough level or RAPA daily dose. *P ⫽ 0.01; **P ⫽ 0.008.
ity of intersubject comparisons. Obviously, the burden of this limitation is increased further in patients switched to RAPA therapy from different immunosuppressive regimens. We explored the ability of RAPA to inhibit P70S6K phosphorylation not only in the basal state, but also after activation of the mTOR cascade in PBMCs by using insulin15,21-23 in an attempt to simulate in vivo the activation of intracellular signaling critical for immune response. Long-term RAPA treat-
ment strongly inhibited both basal and insulinstimulated P70S6K phosphorylation (Fig 3), but inhibition of the target kinase did not show a significant correlation with either RAPA trough level or RAPA daily dose (Fig 4), as already suggested.8,10 Importantly, a single patient showed a stable relationship between RAPA blood concentration and percentage of inhibition of P70S6K activation over time (Fig 5), as opposed to intersubject variability.
Table 2. P70S6K Phosphorylation and Rapamycin Trough Level and Daily Dose in Group II Start of Study (rapamycin ⫹ tacrolimus)
Patient No.
Trough (ng/mL)
Daily Dose (mg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
3.7 3 9.7 6 7.5 5.6 7.6 8.7 4.7 4.4 3.8 5.2 3.5 3.1 8.9 5.3
3 1.5 2.5 2.5 3 2.5 2 2.5 2.5 4 1 2 3 1.5 2 2
Basal P70 (AU)
S6K
1,412.338 1,223.526 1,002.481 1,927.409 1,278.203 4,825.749 4,740.157 1,723.992 4,830.189 3,828.321 2,997.653 5,129.808 1,412.338 1,223.526 1,002.481 1,927.409
Stim P70 (AU)
End of Study (rapamycin alone) S6K
4,867.424 3,745.597 3,712.47 6,059.613 5,147.872 8,178.354 6,785.438 7,675.329 8,161.838 6,543.837 5,518.403 10,385.409 4,867.424 3,745.597 3,712.47 6,059.613
Trough (ng/mL)
Daily Dose (mg)
Basal P70S6K (AU)
Stim P70S6K (AU)
7.9 9.3 8.5 11.4 7.4 8.1 10.4 7.7 6.9 10.1 7 7.5 8.6 9.7 8.1 12.5
2 3.5 1 2 2 4.5 1 5 3 7 2 3 2 4 1 2
1,301.02 774.723 773.842 1,694.635 750.746 2,359.29 2,123.434 1,668.116 5,936.913 2,017.88 1,905.254 4,231.774 1,301.02 774.723 673.842 1,594.635
1,513.605 1,125.984 847.913 1,919.161 872.69 3,941.083 2,809.224 2,636.107 7,380.675 3,275.194 2,819.986 6,812.849 1,513.605 1,125.984 847.913 1,919.161
Note: In each case, phosphorylated P70S6K level is normalized to total P70S6K. Abbreviation: Stim P70S6K, insulin-stimulated phosphorylation of P70S6K.
324
We cannot fully explain the lack of correlation between RAPA trough concentration and degree of inhibition of P70S6K activation. Presumably, it might reflect differential sensitivity of the mTORraptor complex to inhibition by RAPA. In vitro, intrinsic sensitivity to RAPA may vary among different cell lines, even of several orders of magnitude, because of either genetic or epigenetic mechanisms.24 Next, the proportion of the drug that distributes to lymphocytes is only 1% of the entire blood concentration.25 Consequently, small differences in drug proportioning among blood cells that are not reflected by wholeblood trough levels might have a relevant biological impact on the target kinase in PBMCs. Finally, inhibition of P70S6K phosphorylation in PBMCs might correlate with peak RAPA blood levels or 24-hour area under the curve of RAPA absorption, rather than with drug predose concentration. Regardless of the mechanism(s), this finding would question adjustments of RAPA dosage based exclusively on trough levels of the mTOR inhibitor and emphasizes the risk of inappropriate immunosuppression in an individual patient despite the current downsizing of the RAPA therapeutic window.26-28 Our study suggests that the calcineurin inhibitor tacrolimus significantly antagonizes inhibition of P70S6K phosphorylation by RAPA in vivo. In vitro cellular assays initially showed that RAPA and tacrolimus acted as selective reciprocal antagonists in murine T cells, but only in 50to 1,000-fold molar excess.29 However, more recently, it was shown that the magnitude of inhibition of human lymphocyte function slightly increased (if at all) only when tacrolimus concentrations of 10 nmol/L or greater (ⱖ8.2 ng/mL) were combined with equimolar concentrations of RAPA compared with the magnitude of inhibition of lymphocyte function after single drug use.7 The antagonism of immunosuppression is considered to reflect a competition of both drugs to bind FKBP-12, as well as the similar dissociation constants of tacrolimus and RAPA for their common intracellular-binding protein.1 Nonetheless, the assumption of a large excess of intracellular FKBP-12 has led to deny clinical relevance to the antagonism of RAPA and tacrolimus.1 However, additional studies of isolated human PBMCs suggested that the active amount of FKBP-12 limited the immunosuppressive effects
Leogrande et al
of tacrolimus at high concentrations and contradicted the prevailing assumption that immunophilins are abundant and not limiting for tacrolimus activity.30 The clinical relevance of these findings cannot be extrapolated from the present study, although they may suggest some caution in the adoption of subtherapeutic doses of tacrolimus and RAPA when used in combination. In this context, it may be worth mentioning that a large retrospective observational study recently reported significantly worse renal allograft survival in renal transplant recipients administered tacrolimus plus RAPA compared with tacrolimus plus mycophenolate mofetil, particularly for those with higher risk transplants.31 In addition, 2 randomized prospective clinical trials described a trend toward worse graft function and more acute rejection episodes in patients treated with tacrolimus plus RAPA versus tacrolimus plus mycophenolate mofetil, using conventional equimolar dosages of the calcineurin inhibitor and the mTOR inhibitor.32,33 We are aware of some major limitations of the present study. First, we acknowledge that RAPA exerts its antiproliferative and immunosuppressive activities through the modulation of several intracellular pathways lying downstream of mTOR in addition to P70S6K (Fig 1). Therefore, larger studies addressing the correlation of P70S6K activity with renal allograft outcome are required to assess the clinical relevance of the biological marker tested here. Next, the need for in vivo administration of insulin would restrict the assay to patients without diabetes. In addition, the requirement of relatively large amounts of blood (at least 20 mL) makes the assay not suitable for small pediatric patients. In conclusion, we show here that: (1) longterm RAPA treatment causes strong inhibition of basal and mitogen-stimulated P70S6K phosphorylation in PBMCs from renal transplant recipients, with a potency that varies largely among patients and fails to correlate with RAPA trough levels (IC50 range, 3.14 to 12.14 ng/mL); and (2) the combination of tacrolimus and RAPA seemingly limits the inhibitory effect of the latter drug on the activation of P70S6K phosphorylation. ACKNOWLEDGEMENTS Support: None. Financial Disclosure: None.
P70S6 Kinase Inhibition and Rapamycin Efficacy REFERENCES 1. Sehgal SN: Sirolimus: Its discovery, biological properties, and mechanism of action. Transplant Proc 35:SS-S14, 2003 (suppl 3A) 2. MacDonald A, Scarola J, Burke JT, Zimmerman JJ: Clinical pharmacokinetics and therapeutic drug monitoring of sirolimus. Clin Ther 22:SB101-SB121, 2000 (suppl B) 3. Kahan BD, Napoli KL, Kelly PA, et al: Therapeutic drug monitoring of sirolimus: Correlations with efficacy and toxicity. Clin Transplant 14:97-109, 2000 4. Yatscoff RW, Gallant H: P70 S6 kinase assay: A pharmacodynamic monitoring strategy for rapamycin; Assay development. Transplant Proc 28:3058-3061, 1996 5. Ferron GM, Jusko WJ: Species- and gender-related differences in cyclosporine/prednisolone/sirolimus interactions in whole blood lymphocyte proliferation assays. J Pharmacol Exp Ther 286:191-200, 1998 6. Diaz-Romero J, Vogt G, Weckbecker G: Coexpression of CD4 and CD8alpha on rat T-cells in whole blood: A sensitive marker for monitoring T-cell immunosuppressive drugs. J Immunol Methods 254:1-12, 2001 7. Barten MJ, Dhein S, Chang H, et al:Assessment of immunosuppressive drug interactions: Lymphocyte function in peripheral human blood. J Immunol Methods 283:99-114, 2003 8. Peralba JM, deGraffenried L, Friedrichs W, et al: Pharmacodynamic evaluation of CCI-779, an inhibitor of mTOR, in cancer patients. Clin Cancer Res 9:2887-2892, 2003 9. Boulay A, Zumstein-Mecker S, Stephan C, et al: Antitumor efficacy of intermittent treatment schedules with the rapamycin derivative RAD001 correlates with prolonged inactivation of ribosomal protein S6 kinase 1 in peripheral blood mononuclear cells. Cancer Res 64:252-261, 2004 10. Hartmann B, Schmid G, Graeb C, et al: Biochemical monitoring of mTOR inhibitor-based immunosuppression following kidney transplantation: A novel approach for tailored immunosuppressive therapy. Kidney Int 68:2593-2598, 2005 11. Di Paolo S, Teutonico A, Ranieri E, Gesualdo L, Schena FP: Monitoring antitumor efficacy of rapamycin in Kaposi sarcoma. Am J Kidney Dis 49:462-470, 2007 12. Li HL, Davis W, Pure E: Suboptimal cross-linking of antigen receptor induces Syk-dependent activation of p70S6 kinase through protein kinase C and phosphoinositol 3-kinase. J Biol Chem 274:9812-9820, 1999 13. Racusen LC, Solez K, Colvin RB, et al: The Banff 97 working classification of renal allograft pathology. Kidney Int 55:713-723, 1999 14. Teutonico A, Schena PF, Di Paolo S: Glucose metabolism in renal transplant recipients: Effect of calcineurin inhibitor withdrawal and conversion to sirolimus. J Am Soc Nephrol 16:3128-3135, 2005 15. Di Paolo S, Teutonico A, Leogrande D, Capobianco C, Schena PF: Chronic inhibition of mTOR signaling downregulates IRS-1/2 and AKT activation: A crossroad between cancer and diabetes? J Am Soc Nephrol 17:2236-2244, 2006 16. Stallone G, Di Paolo S, Schena A, et al: Addition of sirolimus to cyclosporine delays the recovery from delayed graft function but does not affect 1-year graft function. J Am Soc Nephrol 15:228-233, 2004 17. Hay N, Sonenberg N: Upstream and downstream of mTOR. Genes Dev 18:1926-1945, 2004
325 18. Cattaneo D, Perico N, Gaspari F: Assessment of sirolimus concentrations in whole blood by high-performance liquid chromatography with ultraviolet detection. J Chromatogr B Analyt Technol Biomed Life Sci 774:187-194, 2002 19. Jones K, Johnston A, Holt DW: Proficiency-testing issues relating to sirolimus. Clin Ther 22:SB122-SB132, 2000 (suppl B) 20. Kuo CJ, Chung J, Fiorentino DF, Flanagan WM, Blenis J, Crabtree GR: Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature 358:70-73, 1992 21. Moule SK, Denton RM: Multiple signaling pathways involved in the metabolic effects of insulin. Am J Cardiol 80:41A-49A, 1997 22. Renard E, Grigorescu F, Lavabre C, Kahn CR: Insulindependent phosphatidylinositol 3=-kinase activity co-precipitates with insulin receptor in human circulating mononuclear cells. Biochem Biophys Res Commun 209:234-241, 1995 23. Stentz FB, Kitabchi AE: De novo emergence of growth factor receptors in activated human CD4⫹ and CD8⫹ T lymphocytes. Metabolism 53:117-122, 2004 24. Huang S, Bjornsti MA, Houghton PJ: Rapamycins: Mechanism of action and cellular resistance. Cancer Biol Ther 2:222-232, 2003 25. Trepanier DJ, Gallant H, Legatt DF,Yatscoff RW: Rapamycin: Distribution, pharmacokinetics and therapeutic range investigations: An update. Clin Biochem 31:345-351, 1998 26. Flechner SM, Goldfarb D, Modlin C, et al: Kidney transplantation without calcineurin inhibitor drugs: A prospective, randomized trial of sirolimus versus cyclosporine. Transplantation 74:1070-1076, 2002 27. Baboolal K: A phase III prospective, randomized study to evaluate concentration-controlled sirolimus (Rapamune) with cyclosporine dose minimization or elimination at six months in de novo renal allograft recipients. Transplantation 75:1404-1408, 2003 28. Mota A, Arias M, Taskinen EI, et al: Rapamune Maintenance Regimen Trial: Sirolimus-based therapy following early cyclosporine withdrawal provides significantly improved renal histology and function at 3 years. Am J Transplant 4:953-961, 2004 29. Dumont FJ, Melino MR, Staruch MJ, Koprak SL, Fischer PA, Sigal NH: The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells. J Immunol 144:1418-1424, 1990 30. Kung L, Halloran PF: Immunophilins may limit calcineurin inhibition by cyclosporine and tacrolimus at high drug concentrations. Transplantation 70:327-335, 2000 31. Meier-Kriesche HU, Schold JD, Srinivas TR, Howard RJ, Fujita S, Kaplan B: Sirolimus in combination with tacrolimus is associated with worse renal allograft survival compared to mycophenolate mofetil combined with tacrolimus. Am J Transplant 5:2273-2280, 2005 32. Mendez R, Gonwa T, Yang HC, Weinstein S, Jensik S, Steinberg S, for the Prograf Study Group: A prospective, randomized trial of tacrolimus in combination with sirolimus or mycophenolate mofetil in kidney transplantation: Results at 1 year. Transplantation 80:303-309, 2005 33. Ciancio G, Burke GW, Gaynor JJ, et al: A randomized long-term trial of tacrolimus/sirolimus versus tacrolimus/ mycophenolate versus cyclosporine/sirolimus in renal transplantation: three-year analysis. Transplantation 81:845-852, 2006
R
apamycin (RAPA) is a novel immunosuppressive drug recently approved for use in renal transplant recipients. RAPA penetrates the plasma membrane and binds to FK-binding protein 12 (FKBP-12) to form a complex that binds
From the 1Department of Emergency and Organ Transplants, Division of Nephrology, Dialysis and Transplantation, University of Bari, Policlinico, Bari; 2Department of Biomedical Sciences, Section of Clinical Pathology; and 3 Department of Biomedical Sciences, Division of Nephrology, University of Foggia, Foggia, Italy. Received January 26, 2007. Accepted in revised form May 14, 2007. Originally published online as doi: 10.1053/j.ajkd.2007.05.002 on July 9, 2007. *Present address: Division of Nephrology and Dialysis, Hospital “Dimiccoli,” Barletta, Italy Address correspondence to Salvatore Di Paolo, MD, Division of Nephrology, Hospital “Dimiccoli,” ASL BAT, Viale Ippocrate, 1-Barletta, Italy. E-mail: [email protected] © 2007 by the National Kidney Foundation, Inc. 0272-6386/07/5002-0017$32.00/0 doi:10.1053/j.ajkd.2007.05.002 314
to the mammalian target of RAPA (mTOR).1 This interaction causes dephosphorylation and inactivation of P70 ribosomal S6 kinase (P70S6K), which, when activated, stimulates protein synthesis and cell-cycle progression (Fig 1).1 Both animal and clinical studies showed a relationship between RAPA trough concentrations and graft outcome.1-3 Nonetheless, dose adjustments for such critical-dose drugs as RAPA that rely only on measuring blood concentrations may not necessarily correlate with pharmacological effects of the drugs on immune cells. Thus, identification of molecular markers enabling the assessment and follow-up of biological effects of RAPA may help optimize drug therapy and evaluate new immunosuppressive multidrug regimens. Previous studies investigated the immunosuppressive properties of RAPA by using in vitro or ex vivo experimental models.4-7 Thereafter, a few investigations suggested that the pharmacodynamic effects of RAPA derivatives could be
American Journal of Kidney Diseases, Vol 50, No 2 (August), 2007: pp 314-325
P70S6 Kinase Inhibition and Rapamycin Efficacy
315
Figure 1. A simplified model of the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K)-AKT pathways implicated in the control of T-cell proliferation. Engagement of the T-cell receptor (TCR) and such costimulatory receptors as CD28 elicits expression and secretion of the T-cell growth factor interleukin 2 (IL-2) and expression of its high-affinity receptor (IL-2R), rendering the cells competent for IL-2– driven proliferation. The TCR and CD28 also can control cell-cycle progression independently of IL-2 by direct activation of the PI3K/AKT/mTOR pathway. Similarly, various peptide growth factors, such as insulin, bind to specific cell-surface receptors (I-R) and thereby activate the PI3K/AKT/mTOR pathway. The GL-raptor-mTOR complex controls messenger RNA (mRNA) translation through phosphorylation of P70S6K and eIF4E-binding protein 1 (4EBP1). Activation of P70S6K leads in turn to phosphorylation of the 40S ribosomal protein S6, whereas phosphorylation of 4EBP1 releases the eukaryotic initiation factor 4E (eIF4E) to restore cap-dependent translation. In addition, raptor-mTOR facilitation of G1 to S cell-cycle transition is mediated, at least in part, by the increased translation of mRNAs encoding positive regulators of cell-cycle progression, such as cyclin D3, cyclin E, and c-Myc, and by decreased translation of negative regulators thereof, such as the cyclin-dependent kinase inhibitor p27kip1. Rapamycin (RAPA) binds to FK-binding protein 12 (FKBP-12) and leads to cell-cycle arrest in G1 phase through the inhibition of all these effects of raptor-mTOR, but mainly blocking the phosphorylation of P70S6K and 4EBP1.
316
Leogrande et al
determined reliably in vivo by monitoring P70S6K activity in peripheral-blood mononuclear cells (PBMCs) of patients with cancer.8,9 Next, Hartmann et al10 described pronounced inhibition of basal P70S6K phosphorylation in PBMCs isolated from RAPA-treated kidney transplant recipients and suggested that the phosphorylation status of the kinase can provide more relevant information than RAPA trough levels to prevent acute allograft rejection. Finally, we recently showed that monitoring P70S6K phosphorylation can help predict and monitor regression of cancer lesions in renal transplant recipients with Kaposi sarcoma who were converted to RAPA therapy and possibly adjust the biologically active doses of the mTOR inhibitor.11 The present study is designed with the following aims: (1) to determine whether long-term administration of RAPA in vivo is associated with inhibition of basal and stimulated P70S6K phosphorylation, as shown in vitro; and (2) to assess whether RAPA blood trough concentration, the commonly used index to monitor drug dosing, correlates with degree of P70S6K phosphorylation (ie, activation)12 in kidney transplant recipients on maintenance immunosuppression therapy with RAPA. As a secondary aim, we also investigated possible drug interactions between RAPA and the calcineurin inhibitor tacrolimus in patients treated using a combination of the 2 immunosuppressive drugs. METHODS Patients Two different groups of kidney transplant recipients were investigated.
Group I Starting January 2002 to December 2004, all patients who received a kidney transplant, were 12 to 60 months after transplantation, had stable graft function and serum creatinine levels less than 2.5 mg/dL (⬍221 mol/L), and were in treatment with cyclosporine A microemulsion (CsA), mycophenolate mofetil (1 to 2 g/d), and low-dose steroids (prednisone, 2.5 to 5 mg/d) were offered a protocol graft biopsy. Patients for whom the histological diagnosis of chronic allograft nephropathy13 was made were requested to be converted to RAPA therapy without further modification of the remaining immunosuppressive therapy. Exclusion criteria were age younger than 18 or older than 60 years, histological evidence of recurrent or de novo renal disease, diagnosis of diabetes at any time in their clinical history, significant coexisting severe disease (cardiac, hepatic, or
neoplastic), absolute need for drugs interfering with glucose metabolism,14,15 fasting cholesterol level greater than 300 mg/dL (⬍7.76 mmol/L), and/or triglyceride level greater than 350 mg/dL (⬎3.95 mmol/L). We assessed 232 potential subjects; 102 had already been recruited in different clinical trials, and 22 refused a protocol biopsy. At histological examination, the diagnosis of chronic allograft nephropathy was made in 74 of the remaining 108 patients. Of these, 5 subjects were excluded for the presence of recurrent or de novo glomerular disease, and 19 patients, for diabetes (7 patients), severe hepatic (6 patients) or cardiac (2 patients) disease, or marked hyperlipemia (4 patients). Three transplant recipients underwent investigation for comorbidity, and 8 patients refused to change their immunosuppressive regimen. We eventually recruited 39 patients who started RAPA therapy 12 to 16 hours after the abrupt discontinuation of CsA therapy. All transplant recipients were administered a single oral loading dose of RAPA (0.1 mg/kg), followed by a daily dose of 5 mg. Whole-blood RAPA trough concentration was measured first on day 5 after the conversion, and RAPA daily dose was modified to achieve target trough levels of 8 to 12 ng/mL. All patients were studied immediately before the discontinuation of CsA therapy and 9 to 11 months after the conversion to RAPA therapy.
Group II In the same period at our center, some recipients of suboptimal kidneys16 were randomly assigned to a calcineurin inhibitor–sparing regimen comprised of tacrolimus (target trough level, 6 to 8 ng/mL), RAPA (target trough level, 4 to 8 ng/mL), and low-dose steroids for the first 3 months after grafting, then discontinued tacrolimus therapy and increased RAPA target trough levels to 8 to 12 ng/mL. Collectively, 28 of 93 transplant recipients of suboptimal kidneys were assigned to this regimen; the others entered different clinical trials.16 One patient experienced an acute rejection episode in the first 3 months after engraftment and was converted to full-dose tacrolimus therapy, 2 patients developed posttransplantation diabetes, 1 patient had severe hepatitis C virus–related hepatic disease, 3 patients had marked hypertriglyceridemia, and 1 patient showed progressive graft function deterioration. Therefore, we recruited 20 patients who were examined immediately before and 6 to 8 months after conversion to RAPA-alone therapy. Finally, 7 patients with stable graft function on maintenance therapy with CsA, mycophenolate mofetil, and lowdose steroids were studied twice, with an interval of approximately 6 months, without modification of immunosuppressive drugs, and served as controls to evaluate the spontaneous modification over time of the parameter tested. All patients were asked to give their written informed consent to participate in the study, according to the Guidelines of the Local Ethical Committee.
In Vitro Activation of P70S6K mTOR signaling appears to be regulated downstream of phosphatidylinositol-3-kinase/AKT on T-cell receptor engagement or cell stimulation with lymphokines, growth factors, and hormones (eg, insulin; Fig 1).1,15,17 Preliminary
P70S6 Kinase Inhibition and Rapamycin Efficacy
317
dose–response experiments showed that maximal activation of P70S6K in control PBMCs was achieved 15 minutes after stimulation with 10 nmol/L of insulin, ie, the same order of magnitude of peak serum insulin after intravenous administration. To explore the inhibitory effect of RAPA on P70S6K activation, PBMCs were preincubated for 30 minutes with serial dilutions of RAPA (0 to 10 ng/mL) before the addition of 10 nmol/L of insulin.
quantification, defined as the lowest concentration of drug that could be assayed with a good level of precision and inaccuracy, at 2.5 ng/mL.
In Vivo Activation of mTOR Pathway
Percent inhibition ⫽ [1 ⫺ (Treatment/Pretreatment)] ⫻ 100
To evaluate in vivo the activation of P70S6K and its inhibition by RAPA, 0.1 IU/kg of body weight of soluble insulin was injected intravenously into patients in the fasting state 12 or 24 hours after the last dose of the calcineurin inhibitor or RAPA, respectively.14,15 Blood samples (25 mL) were drawn immediately before and 15 minutes after insulin administration.
Western Blotting Human PBMCs were isolated, lysed, and subjected to blotting as described.11,15 Briefly, 50 g of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene difluoride membrane. The filter was probed with mouse monoclonal antiphospho-P70S6K antibody, raised against a peptide that contained the phosphorylated Ser-411 (Saint Cruz Biotechnologies, Santa Cruz, CA) at 1:100 dilution. Membranes then were washed and incubated with horseradish peroxidase–conjugated goat antimouse immunoglobulin G. To measure total (nonphosphorylated and phosphorylated) kinase, the same membranes were then stripped and immunoblotted with antihuman P70S6K monoclonal antibody raised against a peptide mapping at the carboxy terminus of P70S6K (Saint Cruz Biotechnologies). Cell lysates from each patient were processed on the same gel. The enhanced chemiluminescence system (Amersham Biosciences, Little Chalfont, UK) was used for detection according to the manufacturer’s instructions. The intensity of signals detected by means of enhanced chemiluminescence was quantitated by using densitometric analysis, and results of phospho-P70S6K were expressed in arbitrary units (AU) after normalization to total P70S6K.
Pharmacokinetic Analysis Blood concentration of RAPA was determined by using a high-performance liquid chromatography assay with UV detection, as previously described.18 Application of the method in our laboratory was validated by a reference laboratory that had established an international proficiency testing control system for RAPA.19 The internal standard (32-desmethoxy RAPA) was obtained from Supelco, SigmaAldrich, Milan, Italy. Overall recovery was checked at 5 and 20 ng/mL. The recovery from blood precipitation with zinc sulfate tested was 75.3% ⫾ 3.2% for RAPA and 72.1% ⫾ 2.8% for internal standard, that from reversed-phase extraction was 86.5% ⫾ 3.0% for RAPA and 86.1% ⫾ 2.6% for internal standard. At a 2.5-ng/mL RAPA concentration, within-day and between-day coefficients of variation of the assay were 7.6% and 6.5%, respectively. Inaccuracy of the method was less than 9%. Thus, we set the lower limit of
Pharmacodynamic Analysis In patients in group I, the inhibitory effect of RAPA on P70S6K phosphorylation was calculated using the following formula:
where pretreatment represents the percentage of increase in P70S6K phosphorylation over basal after insulin injection in patients before RAPA therapy, and treatment represents the percentage of increase in P70S6K phosphorylation after longterm treatment with RAPA. The percentage of inhibition of basal P70S6K phosphorylation after RAPA therapy also was calculated. In patients in group II, we were instead interested in exploring the possible interference of tacrolimus on RAPAinduced inhibition of P70S6K activation. To this aim, we compared the percentage of increase in insulin-stimulated P70S6K phosphorylation normalized to RAPA trough level or RAPA daily dose in the presence and absence of the calcineurin inhibitor.
Statistical Analysis Differences between quantitative variables were tested by means of Mann-Whitney U test or Wilcoxon signed-rank test, as appropriate. The relationship between nonparametric variables was tested by using Spearman rank correlation. P less than 0.05 is considered statistically significant. The Statview software package (version 5.0; SAS Institute Inc, Cary, NC) was used for all analyses.
RESULTS
Patients
Thirty of 39 patients in group I completed the study (1 patient developed posttransplantation lymphoproliferative disease, 1 patient progressed to end-stage renal disease, 2 patients developed posttransplantation diabetes, 3 patients had serious unamenable side effects [arthralgias, abdominal pain, and diarrhea] that led to discontinuation of RAPA therapy, 1 patient declined his consent, and 1 patient was lost to follow-up). In group II, 2 patients developed posttransplantation diabetes and 2 patients had serious arthralgias after the conversion to RAPA-alone therapy and were switched to tacrolimus therapy. Consequently, we studied 16 patients. Throughout follow-up, care was taken to avoid any major modification of pharmacological therapy.14,15 In group I, 3 patients had a temporary decrease in mycophenolate mofetil dose at some time during the study because of transient
318
Leogrande et al Table 1. Anthropometric and Laboratory Features of Patients Examined at Study Start and End
Age (y) Sex (M/F) Time from transplantation (mo) Serum creatinine (mg/dL) Daily proteinuria (g/d) Creatinine clearance (mL/min) Triglycerides (mg/dL) Total cholesterol (mg/dL) Leukocytes (/L) Platelets (103/L) CsA/TAC blood levels (ng/mL) CsA/TAC daily dose (mg/kg) RAPA blood levels (ng/mL) RAPA daily dose (mg/kg) MMF dose (mg/d)
Group I
Group II
43.9 ⫾ 12.5 16/14 28.9 ⫾ 13.8
52.0 ⫾ 5.4 12/4 3
Start
End
Start
End
1.46 ⫾ 0.34 0.78 ⫾ 0.24 65.1 ⫾ 21.6 136.7 ⫾ 61.7 204.1 ⫾ 52.5 7,179 ⫾ 2,121 234 ⫾ 56 620.7 ⫾ 147.3 2.38 ⫾ 0.78 — — 1,183 ⫾ 334
1.59 ⫾ 0.42 1.08 ⫾ 0.46 62.9 ⫾ 19.7 201.3 ⫾ 79.5* 228.7 ⫾ 48.8† 6,712 ⫾ 1,814 219 ⫾ 49 — — 9.01 ⫾ 2.21 0.045 ⫾ 0.021 1,150 ⫾ 267
2.17 ⫾ 0.78 1.06 ⫾ 0.63 37.8 ⫾ 11.4 207.1 ⫾ 61.8 212 ⫾ 33.1 6,638 ⫾ 1,531 264 ⫾ 76 7.20 ⫾ 2.65 0.047 ⫾ 0.018 5.66 ⫾ 2.19 0.069 ⫾ 0.035 —
2.10 ⫾ 0.69 1.29 ⫾ 0.84 40.6 ⫾ 10.2 225. ⫾ 127.4 236.4 ⫾ 56.6 7,024 ⫾ 2,016 249 ⫾ 66 — — 8.82 ⫾ 1.62 0.053 ⫾ 0.024 —
Note: Cyclosporine A whole-blood levels were measured 2 hours after the morning dose, rapamycin and tacrolimus levels were obtained by monitoring predose (trough) levels. Results expressed as mean ⫾ SD. To convert serum creatinine in mg/dL to mol/L, multiply by 88.4; creatinine clearance in mL/min to mL/s, multiply by 0.01667; serum cholesterol in mg/dL to mmol/L, multiply by 0.02586; serum triglycerides in mg/dL to mmol/L, multiply by 0.01129. Abbreviations: CsA, cyclosporine A; TAC, tacrolimus; RAPA, rapamycin; MMF, mycophenolate mofetil. *P ⫽ 0.001. †P ⫽ 0.04.
leukopenia, whereas 2 patients permanently decreased their dose from 2 to 1.5 g of mycophenolate mofetil for abdominal pain and leukopenia. No patient studied experienced an acute rejection episode or clinically relevant infection or showed significant modifications in renal function. The main features of patients in each group are listed in Table 1. Most patients showed stable RAPA blood levels over time; the greatest variability was associated with the first 4 to 8 weeks after the start of RAPA therapy.
sponse to the inhibitory effect of the mTOR inhibitor, such that the RAPA concentration required to achieve 50% inhibition of P70S6K activation (IC50) ranged from 0.6 to 4.1 ng/mL among different donors (n ⫽ 4; Fig 2C). Finally, control PBMCs were cultured for 2 to 48 hours in the presence or absence of 200 ng/mL of CsA, then challenged with 10 nmol/L of insulin. The calcineurin inhibitor failed to affect basal or activated phosphorylation of P70S6K, as already reported for AKT activation.15
In Vitro Experiments
Group I In the absence of RAPA, intravenous insulin injection caused a 241.8% ⫾ 94.7% increase in P70S6K phosphorylation over basal levels (Fig 3A and B). Long-term exposure to RAPA was associated with a significant decrease in mean levels of both basal and insulin-stimulated P70S6K phosphorylation without modification of total P70S6K content (Fig 3A and B). Next, we calculated the percentage of change from baseline of phospho-P70S6K, basal and stimulated, in each patient to ensure more accurate assessment of drug-induced changes (Fig 3C).
First we measured the coefficient of variation (CV) of kinase phosphorylation in healthy controls. CVs, calculated as SD/mean of phosphoP70S6K, were 39% for basal and 68% for activated P70S6K phosphorylation (n ⫽ 5). RAPA (10 ng/mL) did not modify basal P70S6K phosphorylation in control PBMCs (not shown). Instead, the mTOR inhibitor significantly downregulated insulin-stimulated phosphorylation of P70S6K in a dose-dependent fashion (Fig 2B and C). However, we observed very large intersubject variability in cell re-
In Vivo Study
P70S6 Kinase Inhibition and Rapamycin Efficacy
319
Figure 2. In vitro activation of P70S6K by insulin and kinase inhibition by rapamycin (RAPA). (A) Time-course of P70S6K phosphorylation by 10 nmol/L of human regular insulin in human peripheral-blood mononuclear cells (PBMCs) isolated from healthy donors. (B, C) Dose-dependent inhibition of insulin-induced activation of P70S6K phosphorylation by RAPA. (B) Representative Western blotting from 1 healthy individual. Kinase activation by 10 ng/mL of interleukin 2 (IL-2) is also represented to the aim of comparison. (C) Quantitation of inhibition of kinase by RAPA (n ⫽ 4). *P ⬍ 0.05; **P ⬍ 0.0001 (versus insulin-treated cells).
RAPA IC50 for mitogen-activated phosphorylation showed large interpatient variability (median, 6.6 ng/mL; range, 3.14 to 12.14 ng/mL). Of note, the degree of inhibition of insulinstimulated P70S6K phosphorylation failed to correlate significantly with RAPA trough levels or daily dose of the mTOR inhibitor (Fig 4). The IC50 of basal P70S6K phosphorylation was strikingly high with a median of 9.35 ng/mL, with very large interpatient variability (3.7 to 21.1 ng/mL). Finally, we wondered whether inhibition of P70S6K phosphorylation would show dose dependence in individual patients. Thus, we reexamined 4 patients after approximately 6 months and found a stable relationship between RAPA trough level and percentage of inhibition of insulinstimulated P70S6K activation, with a CV of per-
centage of inhibition (expected versus measured) of 12.4% (Fig 5). The intra-assay CV for P70S6K phosphorylation was steadily less than 9%. Intraindividual variabilities, tested in 7 controls during a time span of 6 months, were 12.2% and 14.1% for basal and insulin-activated P70S6K phosphorylation, respectively. Instead, the kinase showed large interindividual variability; CVs of basal and stimulated phosphorylation were 47.8% and 38.1%, respectively (n ⫽ 30 patients). Group II Patients in this group were examined first 3 months after engraftment and thereafter were reevaluated 6 months after discontinuation of tacrolimus therapy (Table 1). We sought for a possible interference of tacrolimus on mTOR
320
Leogrande et al
Figure 3. In vivo effect of long-term rapamycin (RAPA) treatment on basal and insulin-stimulated phosphorylation of P70S6K in human peripheral-blood mononuclear cells (PBMCs). Thirty kidney transplant recipients were studied immediately before and 9 to 11 months after discontinuation of cyclosporine A and conversion to RAPA therapy. In vivo activation of the target kinase was obtained by intravenous administration of 0.1 U/kg of human regular insulin. (A) Representative immunoblots: for each patient, all cell lysates were processed on the same gel. (B) Quantification of pP70S6K before and after 9 to 11 months of treatment with RAPA. Results expressed as mean (⫾SD) of absolute values measured in each patient. (C) Change (⌬) in basal and insulin-stimulated P70S6K phosphorylation after long-term RAPA therapy. Data represent mean (⫾SD) of percentage of change from baseline measured in each patient. *P ⬍ 0.0001 versus pretreatment values.
inhibition by RAPA. To this aim, we compared the percentage of increase in insulin-stimulated P70S6K phosphorylation, normalized to either RAPA trough level or RAPA daily dose, in the presence and absence of tacrolimus. As shown in Fig 6, withdrawal of tacrolimus was associated with greater inhibition of kinase activation by RAPA in each individual patient. This suggests that the combination of tacrolimus and RAPA limits the inhibitory effect of the latter drug on mitogen-induced P70S6K phosphorylation.
We also confirmed the lack of relationship between degree of P70S6K phosphorylation in PBMCs and RAPA trough level in this group of patients (Table 2). DISCUSSION
This study shows the ability of RAPA to inhibit in vivo P70S6K activation in PBMCs of renal transplant recipients. Unexpectedly, such inhibition failed to correlate with whole-blood RAPA trough levels. Next, we showed that ta-
P70S6 Kinase Inhibition and Rapamycin Efficacy
321
Figure 4. Lack of a relationship between inhibition of insulin-stimulated phosphorylation of P70S6K and either rapamycin trough level or drug daily dose in patients converted from therapy with cyclosporine to the mTOR inhibitor (group I).
crolimus apparently dampened the inhibitory effect of RAPA on P70S6K phosphorylation. Monitoring RAPA blood levels is recommended to optimize therapy on account of the narrow therapeutic ranges of RAPA, wide intraindividual and interindividual variations in its pharmacokinetics, and potential for significant drug-drug interactions. However, pharmacokinetics cannot account for intersubject variability in the sensitivity to immune suppression by similar blood concentrations of RAPA and cannot measure the biological effect of the drug on immune cells. The selective blockade of the P70S6K acti-
vation cascade by RAPA was shown to efficiently inhibit interleukin 2– and mitogeninduced S phase entry and subsequent T-cell proliferation (Fig 1), resulting in immunosuppression,20 which supports the use of phosphorylation status of P70S6K as a biomarker for mTOR inhibition in RAPA-treated patients. Previously, only a few studies explored the inhibitory effect of RAPA on human lymphocyte function by using in vitro assays in which whole human blood or isolated lymphocytes were exposed to the test drug and mitogens.4,5,7 These investigations reported RAPA IC50 values of at
322
Leogrande et al
Figure 5. Longitudinal assessment of P70S6K activation in 4 individual patients. Results expressed as both (upper panel) absolute values of insulin-stimulated P70S6K phosphorylation and (lower panel) change (⌬) versus pretreatment levels in each patient. Figures over each column represent rapamycin trough levels (nanograms per milliliter) at the time of the assay.
least 30 nmol/L (27.3 ng/mL),5,7 which are definitely greater than the IC50 values reported here and largely greater than the therapeutic range of RAPA. Next, Yatskoff and Gallant4 described an in vitro P70S6K assay on isolated human lymphocytes in which less than 25% inhibition was achieved at a RAPA concentration of 50 ng/mL. In our in vitro model, P70S6K activation was inhibited by RAPA in a dose-dependent fashion, although with large intersubject variability, with an IC50 range of 0.6 to 4.1 ng/mL. A major problem with the use of in vitro studies is that the dose and/or concentration required to inhibit a biological target may not necessarily be similar to the concentration required in vivo. The striking discrepancies among results reported previously apparently corrobo-
rate the difficulty extrapolating findings from in vitro or ex vivo studies to the clinical setting. Hartmann et al10 recently explored the ability of RAPA to inhibit P70S6K basal activation in PBMCs from renal transplant recipients in vivo and suggested that RAPA trough levels of 6 ng/mL or greater adequately suppressed kinase phosphorylation; a cutoff value of 60% phosphorylation relative to controls was highly predictive of acute rejection. However, the study extrapolated the percentage of basal P70S6K phosphorylation inhibition by comparison to the phosphorylation signal of healthy controls. Unfortunately, we and others also found large intersubject variability in basal P70S6K phosphorylation in healthy donors,8,9 similar to immunosuppressed patients,10 and this would greatly limit the reliabil-
P70S6 Kinase Inhibition and Rapamycin Efficacy
323
Figure 6. Insulin-stimulated P70S6K phosphorylation in 16 renal transplant recipients while treated with rapamycin (RAPA) plus low-dose tacrolimus (⫹) and 6 months after conversion to RAPAalone therapy (⫺). Percentage of increase in P70S6K phosphorylation over basal is normalized to either RAPA trough level or RAPA daily dose. *P ⫽ 0.01; **P ⫽ 0.008.
ity of intersubject comparisons. Obviously, the burden of this limitation is increased further in patients switched to RAPA therapy from different immunosuppressive regimens. We explored the ability of RAPA to inhibit P70S6K phosphorylation not only in the basal state, but also after activation of the mTOR cascade in PBMCs by using insulin15,21-23 in an attempt to simulate in vivo the activation of intracellular signaling critical for immune response. Long-term RAPA treat-
ment strongly inhibited both basal and insulinstimulated P70S6K phosphorylation (Fig 3), but inhibition of the target kinase did not show a significant correlation with either RAPA trough level or RAPA daily dose (Fig 4), as already suggested.8,10 Importantly, a single patient showed a stable relationship between RAPA blood concentration and percentage of inhibition of P70S6K activation over time (Fig 5), as opposed to intersubject variability.
Table 2. P70S6K Phosphorylation and Rapamycin Trough Level and Daily Dose in Group II Start of Study (rapamycin ⫹ tacrolimus)
Patient No.
Trough (ng/mL)
Daily Dose (mg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
3.7 3 9.7 6 7.5 5.6 7.6 8.7 4.7 4.4 3.8 5.2 3.5 3.1 8.9 5.3
3 1.5 2.5 2.5 3 2.5 2 2.5 2.5 4 1 2 3 1.5 2 2
Basal P70 (AU)
S6K
1,412.338 1,223.526 1,002.481 1,927.409 1,278.203 4,825.749 4,740.157 1,723.992 4,830.189 3,828.321 2,997.653 5,129.808 1,412.338 1,223.526 1,002.481 1,927.409
Stim P70 (AU)
End of Study (rapamycin alone) S6K
4,867.424 3,745.597 3,712.47 6,059.613 5,147.872 8,178.354 6,785.438 7,675.329 8,161.838 6,543.837 5,518.403 10,385.409 4,867.424 3,745.597 3,712.47 6,059.613
Trough (ng/mL)
Daily Dose (mg)
Basal P70S6K (AU)
Stim P70S6K (AU)
7.9 9.3 8.5 11.4 7.4 8.1 10.4 7.7 6.9 10.1 7 7.5 8.6 9.7 8.1 12.5
2 3.5 1 2 2 4.5 1 5 3 7 2 3 2 4 1 2
1,301.02 774.723 773.842 1,694.635 750.746 2,359.29 2,123.434 1,668.116 5,936.913 2,017.88 1,905.254 4,231.774 1,301.02 774.723 673.842 1,594.635
1,513.605 1,125.984 847.913 1,919.161 872.69 3,941.083 2,809.224 2,636.107 7,380.675 3,275.194 2,819.986 6,812.849 1,513.605 1,125.984 847.913 1,919.161
Note: In each case, phosphorylated P70S6K level is normalized to total P70S6K. Abbreviation: Stim P70S6K, insulin-stimulated phosphorylation of P70S6K.
324
We cannot fully explain the lack of correlation between RAPA trough concentration and degree of inhibition of P70S6K activation. Presumably, it might reflect differential sensitivity of the mTORraptor complex to inhibition by RAPA. In vitro, intrinsic sensitivity to RAPA may vary among different cell lines, even of several orders of magnitude, because of either genetic or epigenetic mechanisms.24 Next, the proportion of the drug that distributes to lymphocytes is only 1% of the entire blood concentration.25 Consequently, small differences in drug proportioning among blood cells that are not reflected by wholeblood trough levels might have a relevant biological impact on the target kinase in PBMCs. Finally, inhibition of P70S6K phosphorylation in PBMCs might correlate with peak RAPA blood levels or 24-hour area under the curve of RAPA absorption, rather than with drug predose concentration. Regardless of the mechanism(s), this finding would question adjustments of RAPA dosage based exclusively on trough levels of the mTOR inhibitor and emphasizes the risk of inappropriate immunosuppression in an individual patient despite the current downsizing of the RAPA therapeutic window.26-28 Our study suggests that the calcineurin inhibitor tacrolimus significantly antagonizes inhibition of P70S6K phosphorylation by RAPA in vivo. In vitro cellular assays initially showed that RAPA and tacrolimus acted as selective reciprocal antagonists in murine T cells, but only in 50to 1,000-fold molar excess.29 However, more recently, it was shown that the magnitude of inhibition of human lymphocyte function slightly increased (if at all) only when tacrolimus concentrations of 10 nmol/L or greater (ⱖ8.2 ng/mL) were combined with equimolar concentrations of RAPA compared with the magnitude of inhibition of lymphocyte function after single drug use.7 The antagonism of immunosuppression is considered to reflect a competition of both drugs to bind FKBP-12, as well as the similar dissociation constants of tacrolimus and RAPA for their common intracellular-binding protein.1 Nonetheless, the assumption of a large excess of intracellular FKBP-12 has led to deny clinical relevance to the antagonism of RAPA and tacrolimus.1 However, additional studies of isolated human PBMCs suggested that the active amount of FKBP-12 limited the immunosuppressive effects
Leogrande et al
of tacrolimus at high concentrations and contradicted the prevailing assumption that immunophilins are abundant and not limiting for tacrolimus activity.30 The clinical relevance of these findings cannot be extrapolated from the present study, although they may suggest some caution in the adoption of subtherapeutic doses of tacrolimus and RAPA when used in combination. In this context, it may be worth mentioning that a large retrospective observational study recently reported significantly worse renal allograft survival in renal transplant recipients administered tacrolimus plus RAPA compared with tacrolimus plus mycophenolate mofetil, particularly for those with higher risk transplants.31 In addition, 2 randomized prospective clinical trials described a trend toward worse graft function and more acute rejection episodes in patients treated with tacrolimus plus RAPA versus tacrolimus plus mycophenolate mofetil, using conventional equimolar dosages of the calcineurin inhibitor and the mTOR inhibitor.32,33 We are aware of some major limitations of the present study. First, we acknowledge that RAPA exerts its antiproliferative and immunosuppressive activities through the modulation of several intracellular pathways lying downstream of mTOR in addition to P70S6K (Fig 1). Therefore, larger studies addressing the correlation of P70S6K activity with renal allograft outcome are required to assess the clinical relevance of the biological marker tested here. Next, the need for in vivo administration of insulin would restrict the assay to patients without diabetes. In addition, the requirement of relatively large amounts of blood (at least 20 mL) makes the assay not suitable for small pediatric patients. In conclusion, we show here that: (1) longterm RAPA treatment causes strong inhibition of basal and mitogen-stimulated P70S6K phosphorylation in PBMCs from renal transplant recipients, with a potency that varies largely among patients and fails to correlate with RAPA trough levels (IC50 range, 3.14 to 12.14 ng/mL); and (2) the combination of tacrolimus and RAPA seemingly limits the inhibitory effect of the latter drug on the activation of P70S6K phosphorylation. ACKNOWLEDGEMENTS Support: None. Financial Disclosure: None.
P70S6 Kinase Inhibition and Rapamycin Efficacy REFERENCES 1. Sehgal SN: Sirolimus: Its discovery, biological properties, and mechanism of action. Transplant Proc 35:SS-S14, 2003 (suppl 3A) 2. MacDonald A, Scarola J, Burke JT, Zimmerman JJ: Clinical pharmacokinetics and therapeutic drug monitoring of sirolimus. Clin Ther 22:SB101-SB121, 2000 (suppl B) 3. Kahan BD, Napoli KL, Kelly PA, et al: Therapeutic drug monitoring of sirolimus: Correlations with efficacy and toxicity. Clin Transplant 14:97-109, 2000 4. Yatscoff RW, Gallant H: P70 S6 kinase assay: A pharmacodynamic monitoring strategy for rapamycin; Assay development. Transplant Proc 28:3058-3061, 1996 5. Ferron GM, Jusko WJ: Species- and gender-related differences in cyclosporine/prednisolone/sirolimus interactions in whole blood lymphocyte proliferation assays. J Pharmacol Exp Ther 286:191-200, 1998 6. Diaz-Romero J, Vogt G, Weckbecker G: Coexpression of CD4 and CD8alpha on rat T-cells in whole blood: A sensitive marker for monitoring T-cell immunosuppressive drugs. J Immunol Methods 254:1-12, 2001 7. Barten MJ, Dhein S, Chang H, et al:Assessment of immunosuppressive drug interactions: Lymphocyte function in peripheral human blood. J Immunol Methods 283:99-114, 2003 8. Peralba JM, deGraffenried L, Friedrichs W, et al: Pharmacodynamic evaluation of CCI-779, an inhibitor of mTOR, in cancer patients. Clin Cancer Res 9:2887-2892, 2003 9. Boulay A, Zumstein-Mecker S, Stephan C, et al: Antitumor efficacy of intermittent treatment schedules with the rapamycin derivative RAD001 correlates with prolonged inactivation of ribosomal protein S6 kinase 1 in peripheral blood mononuclear cells. Cancer Res 64:252-261, 2004 10. Hartmann B, Schmid G, Graeb C, et al: Biochemical monitoring of mTOR inhibitor-based immunosuppression following kidney transplantation: A novel approach for tailored immunosuppressive therapy. Kidney Int 68:2593-2598, 2005 11. Di Paolo S, Teutonico A, Ranieri E, Gesualdo L, Schena FP: Monitoring antitumor efficacy of rapamycin in Kaposi sarcoma. Am J Kidney Dis 49:462-470, 2007 12. Li HL, Davis W, Pure E: Suboptimal cross-linking of antigen receptor induces Syk-dependent activation of p70S6 kinase through protein kinase C and phosphoinositol 3-kinase. J Biol Chem 274:9812-9820, 1999 13. Racusen LC, Solez K, Colvin RB, et al: The Banff 97 working classification of renal allograft pathology. Kidney Int 55:713-723, 1999 14. Teutonico A, Schena PF, Di Paolo S: Glucose metabolism in renal transplant recipients: Effect of calcineurin inhibitor withdrawal and conversion to sirolimus. J Am Soc Nephrol 16:3128-3135, 2005 15. Di Paolo S, Teutonico A, Leogrande D, Capobianco C, Schena PF: Chronic inhibition of mTOR signaling downregulates IRS-1/2 and AKT activation: A crossroad between cancer and diabetes? J Am Soc Nephrol 17:2236-2244, 2006 16. Stallone G, Di Paolo S, Schena A, et al: Addition of sirolimus to cyclosporine delays the recovery from delayed graft function but does not affect 1-year graft function. J Am Soc Nephrol 15:228-233, 2004 17. Hay N, Sonenberg N: Upstream and downstream of mTOR. Genes Dev 18:1926-1945, 2004
325 18. Cattaneo D, Perico N, Gaspari F: Assessment of sirolimus concentrations in whole blood by high-performance liquid chromatography with ultraviolet detection. J Chromatogr B Analyt Technol Biomed Life Sci 774:187-194, 2002 19. Jones K, Johnston A, Holt DW: Proficiency-testing issues relating to sirolimus. Clin Ther 22:SB122-SB132, 2000 (suppl B) 20. Kuo CJ, Chung J, Fiorentino DF, Flanagan WM, Blenis J, Crabtree GR: Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature 358:70-73, 1992 21. Moule SK, Denton RM: Multiple signaling pathways involved in the metabolic effects of insulin. Am J Cardiol 80:41A-49A, 1997 22. Renard E, Grigorescu F, Lavabre C, Kahn CR: Insulindependent phosphatidylinositol 3=-kinase activity co-precipitates with insulin receptor in human circulating mononuclear cells. Biochem Biophys Res Commun 209:234-241, 1995 23. Stentz FB, Kitabchi AE: De novo emergence of growth factor receptors in activated human CD4⫹ and CD8⫹ T lymphocytes. Metabolism 53:117-122, 2004 24. Huang S, Bjornsti MA, Houghton PJ: Rapamycins: Mechanism of action and cellular resistance. Cancer Biol Ther 2:222-232, 2003 25. Trepanier DJ, Gallant H, Legatt DF,Yatscoff RW: Rapamycin: Distribution, pharmacokinetics and therapeutic range investigations: An update. Clin Biochem 31:345-351, 1998 26. Flechner SM, Goldfarb D, Modlin C, et al: Kidney transplantation without calcineurin inhibitor drugs: A prospective, randomized trial of sirolimus versus cyclosporine. Transplantation 74:1070-1076, 2002 27. Baboolal K: A phase III prospective, randomized study to evaluate concentration-controlled sirolimus (Rapamune) with cyclosporine dose minimization or elimination at six months in de novo renal allograft recipients. Transplantation 75:1404-1408, 2003 28. Mota A, Arias M, Taskinen EI, et al: Rapamune Maintenance Regimen Trial: Sirolimus-based therapy following early cyclosporine withdrawal provides significantly improved renal histology and function at 3 years. Am J Transplant 4:953-961, 2004 29. Dumont FJ, Melino MR, Staruch MJ, Koprak SL, Fischer PA, Sigal NH: The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells. J Immunol 144:1418-1424, 1990 30. Kung L, Halloran PF: Immunophilins may limit calcineurin inhibition by cyclosporine and tacrolimus at high drug concentrations. Transplantation 70:327-335, 2000 31. Meier-Kriesche HU, Schold JD, Srinivas TR, Howard RJ, Fujita S, Kaplan B: Sirolimus in combination with tacrolimus is associated with worse renal allograft survival compared to mycophenolate mofetil combined with tacrolimus. Am J Transplant 5:2273-2280, 2005 32. Mendez R, Gonwa T, Yang HC, Weinstein S, Jensik S, Steinberg S, for the Prograf Study Group: A prospective, randomized trial of tacrolimus in combination with sirolimus or mycophenolate mofetil in kidney transplantation: Results at 1 year. Transplantation 80:303-309, 2005 33. Ciancio G, Burke GW, Gaynor JJ, et al: A randomized long-term trial of tacrolimus/sirolimus versus tacrolimus/ mycophenolate versus cyclosporine/sirolimus in renal transplantation: three-year analysis. Transplantation 81:845-852, 2006