RESUMO
BACKGROUND: The once-daily formulation of tacrolimus (TAC(OD)) has been developed to overcome adherence problems. Conversion from the twice-daily TAC (TAC(BID)) formulation to TAC(OD) on a 1:1 basis, however, often leads to a decrease of TAC predose concentrations, which averages â¼15%. Switching between the two TAC formulations may thus influence drug efficacy and necessitates therapeutic drug monitoring. As an additional tool in transplantation diagnostics, phospho-specific flow cytometry was used to study the biological effects of conversion on p38MAPK phosphorylation, a kinase involved in T-lymphocyte activation. METHODS: Stable renal transplant recipients (n = 12), at least 1 year after their transplantation, were converted from TAC(BID) to TAC(OD) on 1:1 mg for mg base. Comedication consisted of mycophenolate mofetil (n = 10) and prednisolone (n = 3). TAC whole-blood predose concentrations were determined by immunoassay before and 3 months after conversion. P38MAPK phosphorylation was measured in T lymphocytes by whole-blood phospho-specific flow cytometry. RESULTS: Three months after conversion, no significant decreases in TAC predose concentrations (C0) were found (P = 0.54), whereas p38MAPK phosphorylation increased with 11.4% (P < 0.05) in CD4 and with 15.6% (P < 0.05) in CD8 T lymphocytes. The TAC C0 during treatment with TAC(BID) correlated inversely with p38MAPK phosphorylation in T lymphocytes (rs = -0.638; P < 0.05). CONCLUSIONS: These results suggest that the measurement of p38MAPK phosphorylation status in T lymphocytes is a sensitive method to determine the biological effects of TAC before and after conversion from TAC(BID) to TAC(OD). This method could be a more sensitive tool for therapeutic drug monitoring of TAC.
Assuntos
Rejeição de Enxerto/prevenção & controle , Imunossupressores/administração & dosagem , Fosforilação/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Tacrolimo/administração & dosagem , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adulto , Idoso , Esquema de Medicação , Monitoramento de Medicamentos/métodos , Feminino , Rejeição de Enxerto/sangue , Rejeição de Enxerto/metabolismo , Humanos , Imunossupressores/sangue , Transplante de Rim/métodos , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/administração & dosagem , Prednisolona/administração & dosagem , Linfócitos T/metabolismo , Tacrolimo/sangue , Transplantados , Adulto JovemRESUMO
INTRODUCTION: Tacrolimus has a large interindividual pharmacokinetic variability, and quantification of its effect is difficult. It is a substrate of ABCB1, an efflux pump expressed more on CD8 T cells than on CD4 T cells. The ABCB1 3435C>T single-nucleotide polymorphism (SNP) has been associated with interindividual differences in ABCB1 activity and may influence drug efficacy. Here the influence of this SNP on the biological effect of tacrolimus was studied. METHODS: Rhodamine (Rh123) efflux was used to study ABCB1 activity, with or without the addition of the ABCB1 inhibitor verapamil. Intracellular interleukin (IL) 2 production in T cells was used to measure the pharmacodynamic effect of tacrolimus after phorbol-12-myristate-13-acetate/ionomycin stimulation of whole blood. In addition, the ABCB1 genotype of 36 tacrolimus-treated renal transplant patients was related to ABCB1 activity and tacrolimus efficacy. RESULTS: The mean Rh123 efflux was higher in CD8 T cells compared with CD4 T cells: 40% versus 19% of cells, respectively (P < 0.001). Verapamil almost completely blocked Rh123 efflux (to 1.8% of CD4 T cells and 0.5% of CD8 T cells), whereas tacrolimus did not change Rh123 efflux. Tacrolimus 10 ng/mL reduced the production of IL-2 in CD4 and CD8 T cells by 28.9% and 45.4% (P < 0.05). Tacrolimus-mediated inhibition of IL-2 was enhanced by verapamil (P < 0.05). This effect on tacrolimus pharmacodynamics was associated with ABCB1 3435C>T SNP in renal transplant patients: verapamil reduced the percentage of IL-2-producing CD4 and CD8 T cells by 14% and 22% in patients with the CC genotype (P < 0.05) but not in patients with the TT genotype. Moreover, the ratio of tacrolimus C0 over the percent of IL-2-producing CD8 T cells in CC genotype patients was significantly higher compared with TT genotype patients (P < 0.05), showing a smaller pharmacodynamic effect in CC genotype patients. CONCLUSION: The ABCB1 3435C>T SNP influences ABCB1 activity of T cells and the pharmacodynamic effect of tacrolimus in kidney transplant patients.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Imunossupressores/farmacologia , Tacrolimo/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Genótipo , Rejeição de Enxerto/prevenção & controle , Humanos , Imunossupressores/farmacocinética , Interleucina-2/genética , Transplante de Rim/métodos , Polimorfismo de Nucleotídeo Único , Tacrolimo/farmacocinéticaRESUMO
The calcineurin inhibitor, tacrolimus (TAC), inhibits the protein phosphatase activity of calcineurin, leading to suppression of the nuclear translocation of NFAT and inhibition of T cell activation. Apart from NFAT also the transcription factor NF-κB plays a key functional role in T cell activation. Therefore, blockade of the NF-κB activation cascade by immunosuppressive drugs prevents immune activation. Here we studied whether TAC blocks NF-κB activation in peripheral human T cells. After anti-CD3/CD28-activation of T cells from healthy volunteers, NF-κB (p65) phosphorylation was measured by flow cytometry in CD3+ T cells, CD4+ helper T cells and CD8+ cytotoxic T cells in the absence and presence of TAC 10 ng/mL, sotrastaurin 500 nM (positive control) and mycophenolic acid 10 µg/mL (negative control; n = 6). NF-κB transcriptional activity was measured by ELISA and intracellular TNFα protein, a downstream target, was measured by flow cytometry to assess the functional consequences of NF-κB blockade. Anti-CD3/28-activation induced NF-κB phosphorylation in CD3+ T cells, CD4+ T cells and CD8+ T cells by 34% (mean), 38% and 30% resp. (p<0.01). Sotrastaurin inhibited NF-κB activation in the respective T cell subsets by 93%, 95% and 86% (p<0.01 vs. no drug), while mycophenolic acid did not affect this activation pathway. Surprisingly, TAC also inhibited NF-κB phosphorylation, by 55% (p<0.01) in CD3+ T cells, by 56% (p<0.01) in CD4+ T cells and by 51% in CD8+ T cells (p<0.01). In addition, TAC suppressed NF-κB DNA binding capacity by 55% (p<0.05) in CD3+ T cells and TNFα protein expression was inhibited in CD3+ T cells, CD4+ T cells and CD8+ T cells by 76%, 71% and 93% resp. (p<0.01 vs. no drug), confirming impaired NF-κB signaling. This study shows the suppressive effect of TAC on NF-κB signaling in peripheral human T cell subsets, measured at three specific positions in the NF-κB activation cascade.
Assuntos
Imunossupressores/farmacologia , NF-kappa B/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Tacrolimo/farmacologia , Inibidores de Calcineurina , Células Cultivadas , Citocinas/metabolismo , Humanos , Fosforilação , Ligação Proteica , Pirróis/farmacologia , Quinazolinas/farmacologia , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Cytokines of the IL-2 receptor family act via activation of the JAK-STAT (janus tyrosine kinase-signal transducer and activator of transcription) signaling pathway. These cytokines are pivotal for the development and function of lymphocyte subsets involved in the immune response after organ transplantation including T, B and natural killer cells. The new small drug molecule and JAK1/3 inhibitor, tofacitinib, is currently being tested in phase II and III clinical trials for rheumatoid arthritis, psoriasis and in organ transplantation. This agent specifically targets the JAK-STAT signaling pathway. Here we discuss phosphospecific flow cytometry as a novel tool to monitor the JAK-STAT signaling pathway in kidney transplant patients and speculate that through the use of this pharmacodynamic tool the efficacy of immunosuppressive drugs can be assessed enabling optimization of the immunosuppressive therapy for individual transplant patients.
Assuntos
Monitoramento de Medicamentos , Citometria de Fluxo , Imunossupressores/farmacocinética , Janus Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Fatores de Transcrição STAT/metabolismo , Animais , Humanos , Imunossupressores/farmacologia , Janus Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição STAT/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Organ transplant recipients frequently suffer from toxicity or from lack of efficacy of immunosuppressive drugs, which can be attributed to individual variations in drug sensitivity. This problem can be resolved by applying pharmacodynamic monitoring that focuses on measuring the biological effects of drugs. Here we discuss the new technique called phospho-specific flow cytometry to monitor the activity of intracellular immune signaling pathways at the single-cell level in whole blood samples. Through this tool the efficacy of immunosuppressive medication can be assessed, novel targets can be identified, and differences in drug sensitivity between cells and patients can be clarified.
RESUMO
BACKGROUND: Tacrolimus (TAC), the cornerstone of immunosuppressive therapy after solid organ transplantation, inhibits calcineurin activation. Despite pharmacokinetic monitoring, patients frequently experience toxicity or lack of efficacy, which could be prevented by pharmacodynamic monitoring. In Jurkat T-cell lines, it has been shown that TAC, in addition to calcineurin, inhibits the p38 mitogen-activated protein kinase (MAPK) pathway, which is important in T-cell activation and is therefore a potential drug-specific biomarker. We studied whether TAC inhibits p38 MAPK signaling in primary human T cells and ex vivo in healthy volunteers and kidney transplant recipients. METHODS: Phorbol-12-myristate-13-acetate/ionomycin-induced MAPK signaling was measured by whole-blood phosphospecific flow cytometry. RESULTS: In vitro, 10-ng/mL TAC inhibited p38 MAPK phosphorylation by a mean of 27% in CD3, 26% in CD4, and 34% in CD8 T cells (P<0.01 compared with baseline). In healthy adults (n=4), 2 hr after a single oral dose of 10-mg TAC, the p38 MAPK activation was inhibited by 35% in CD3, CD4, and CD8 T cells (P<0.05 compared with baseline). In kidney transplant recipients (n=24), TAC predose concentrations (range, 3.2-10.5 ng/mL) were inversely correlated with p38 MAPK activation in CD3, CD4, and CD8 T cells (r=0.51, 0.34, and 0.37, respectively; P<0.01). CONCLUSIONS: TAC inhibits activation of the MAPK pathway in a dose-dependent manner in kidney transplant patients and may be a potential marker for immune monitoring.
Assuntos
Citometria de Fluxo/métodos , Rejeição de Enxerto/tratamento farmacológico , Transplante de Rim , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Tacrolimo/administração & dosagem , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adulto , Carcinógenos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Imunossupressores/administração & dosagem , Células Jurkat , Sistema de Sinalização das MAP Quinases/imunologia , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/sangue , Fosforilação/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
BACKGROUND: The common γ-chain (γ(c)) cytokines signal through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and play pivotal roles in lymphocyte activation. We investigated the effect of immunosuppressive drugs targeting this pathway, the JAK inhibitor tofacitinib (CP-690,550) and the anti-interleukin (IL)-2R antibody basiliximab, as part of a phase 2 study. METHODS: After whole-blood activation with the γ(c) cytokines IL-2, IL-7, and IL-15, STAT5 phosphorylation was determined in T cells of de novo kidney transplantation patients treated with tofacitinib/basiliximab (n=5), calcineurin inhibitor (CNI) (cyclosporine A)/basiliximab (n=4) or CNI (tacrolimus)-based immunosuppression (n=6). The IC(50) for phosphorylated STAT (P-STAT) 5 inhibition by tofacitinib was determined in cytokine-activated CD4(+) and CD8(+) T cells from healthy individuals (n=4). RESULTS: IC(50) was 26, 72, and 37 ng/mL for IL-2, IL-7, and IL-15 activation, in CD4(+) T cells, respectively; and 35, 61, and 76 ng/mL for IL-2, IL-7, and IL-15 activation, in CD8(+) T cells, respectively. In kidney transplantation patients, 7 days after starting tofacitinib/basiliximab treatment, cytokine-induced P-STAT5 was inhibited in CD4(+) T cells (92% for IL-2 activation, 60% for IL-7, and 75% for IL-15), which persisted for the 2-month study period. In contrast, CNI/basiliximab treatment did not affect IL-7-activated or IL-15-activated P-STAT5; only IL-2-activated P-STAT5 was reduced by 77% on day 7 and recovered to pretreatment levels within 2 months. CD8(+) T cells showed a comparable profile to CD4(+) T cells. P-STAT5 was not inhibited in CNI-treated control patients. CONCLUSIONS: Tofacitinib therapy strongly inhibits γ(c) cytokine-induced JAK/STAT5 activation, whereas basiliximab suppresses IL-2-stimulated activation only. Pharmacodynamic monitoring offers a unique tool to evaluate the biologic effects of immunosuppressive drugs.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunossupressores/uso terapêutico , Janus Quinases/antagonistas & inibidores , Transplante de Rim/imunologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Receptores de Interleucina-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , Basiliximab , Ciclosporina/uso terapêutico , Quimioterapia Combinada , Humanos , Interleucina-15/metabolismo , Interleucina-2/metabolismo , Interleucina-7/metabolismo , Janus Quinases/metabolismo , Contagem de Linfócitos , Linfócitos/enzimologia , Linfócitos/imunologia , Países Baixos , Fosforilação , Piperidinas , Receptores de Interleucina-2/imunologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tacrolimo/uso terapêutico , Fatores de Tempo , Resultado do TratamentoRESUMO
Some clinicians advocate using metered-dose inhaler (MDI) spacers prepared from common household bottles. We evaluated the in vitro aerosol characteristics of fluticasone from the MDI alone and through spacers fashioned from 237-, 500-, 1000-, and 1500-mL polyethylene terephlate plastic cold drink bottles using a cascade impactor. Ten 110-microg actuations of fluticasone were sampled into the impactor during each of five runs with each configuration. The primary outcomes were the respirable dose (i.e., quantity aerosol 1.1-4.7 microm in size) and the quantity trapped in the oropharynx. The mean fluticasone respirable dose from the MDI alone (46.2 microg/actuation) was no different (p > 0.05) compared with the same MDI mated to any of the bottle spacers, regardless of volume. The mean quantity of fluticasone trapped in the oropharynx from the MDI alone (39.2 microg/actuation) was greater (p < 0.001) than the same MDI mated with any bottle spacer (range 1.5-3.5 microg/actuation). These data suggest that any of the bottle spacers tested would be an acceptable method to decrease the quantity of fluticasone that would deposit onto the oropharynx and thereby diminish the risk of topical adverse effects. Among the range of volumes tested, there was no relationship between spacer volume and respirable dose of fluticasone.
Assuntos
Androstadienos/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Espaçadores de Inalação , Inaladores Dosimetrados , Desenho de Equipamento , Fluticasona , Humanos , Orofaringe , PolietilenotereftalatosRESUMO
BACKGROUND: Previous in vitro aerosol deposition experiments indicate that the corticosteroid respirable dose from a metered-dose inhaler (MDI) can vary by threefold depending on the specific valved holding chamber (VHC) MDI combination. OBJECTIVE: We compared in vitro aerosol deposition from a fluticasone propionate MDI (Flovent, GlaxoSmithKline, Research Triangle Park, NC) to that of the same MDI used in combination with two VHCs (EasiVent, Dey, Napa, Ca; and AeroChamber-Plus, Monaghan Medical Corp, Plattsburgh, NY) to evaluate how these VHCs affect the respirable dose of fluticasone. METHODS: The respirable dose (aerosol particles 1 to 5 microm in size) of fluticasone was determined by sampling 5 x 110-microg actuations from each configuration (MDI alone, MDI plus AeroChamber-Plus, and MDI plus EasiVent) in multiples of ten using a well established, in vitro cascade impactor method. Fluticasone aerosol was washed from individual impactor stages with 50% methanol and quantified via ultraviolet high-pressure liquid chromatography. Differences among outcomes were determined using analysis of variance. RESULTS: Mean respirable dose from AeroChamber-Plus (47.9 +/- 6.9 microg/actuation) was not different (P > 0.05) from that produced by the MDI alone (50.3 +/- 2.2 microg/actuation). EasiVent respirable dose (27.0 +/- 3.6 microg/actuation) was less than that produced by either the AeroChamber-Plus or the MDI alone (P < 0.001). CONCLUSIONS: VHCs do not display equivalent in vitro performance with a fluticasone MDI. If a patient needs a VHC, clinicians should use available in vitro performance information to aid in selecting the best device.